Antibodies against clostridium difficile

ABSTRACT

Compositions and methods for the treatment or prevention of  Clostridium difficile  infection in a subject are provided. The compositions comprise antibodies to  Clostridium difficile  toxin B. The methods provide for administering the antibodies to a subject in an amount effective to reduce or eliminate or prevent relapse from  Clostridium difficile  bacterial infection.

FIELD

The invention relates to monoclonal antibodies to Clostridium difficile toxin B. The invention further relates to compositions and methods for the diagnosis, treatment or prevention of infection by the bacteria, Clostridium difficile, in a vertebrate subject. Methods are provided for administering antibodies to the vertebrate subject in an amount effective to reduce, eliminate, or prevent relapse from infection.

BACKGROUND

Clostridium difficile (C. difficile) is a common nosocomial pathogen and a major cause of morbidity and mortality among hospitalized patients throughout the world. Kelly et al., New Eng. J. Med., 330:257-62, 1994. The increased use of broad spectrum antibiotics and the emergence of unusually virulent strains of C. difficile have lead to the idea that immunotherapies may be well suited to reduce disease and death associated with this bacterium. C. difficile has few traditional antibiotic options and frequently causes a recurring disease (25% of cases). Even with medical intervention, C. difficile claims about 20,000 lives in the USA alone per year and causes around 500,000 confirmed infections. Recently, more virulent strains of C. difficile have emerged that produce elevated levels of toxin such as the B1/NAP1/027 strain, which also has a decreased susceptibility to metronidazole. Outbreaks of C. difficile have necessitated ward and partial hospital closure due to the persistence of the spores that facilitate the spread of the disease. With the increasing elderly population and the changing demographics of the population, C. difficile is set to become a major problem in the 21^(st) century. The spectrum of C. difficile disease ranges from asymptomatic carriage to mild diarrhea to fulminant pseudomembranous colitis.

C. difficile has a dimorphic lifecycle whereby it exists both as an infectious and tough spore form and a metabolically active toxin-producing vegetative cell. C. difficile-associated disease (CDAD) is believed to be caused by the vegetative cells and more specifically the actions of two toxins, enterotoxin toxin A and cytotoxin toxin B. To date, vaccines and immune therapy for C. difficile have focused upon the toxins (A and B), toxoids of A and B, recombinant fragments of A and B, and vegetative cell surface layer proteins (SLPAs).

Toxin B (TcdB, ˜269 kDa) is an approximately 2366 residue single polypeptide toxin encoded on a C. difficile pathogenicity locus (PaLoc) that also includes genes for two regulators (TcdC and TcdR) of toxin expression, a putative holin (TcdE), and Toxin A (TcdA). TcdB has at least four functional domains that contribute to cell entry and glucosylation of small-GTPases within the cytosol of the cell. TcdB's glucosyltransferase domain is included in the first 543 residues of the toxin and is the biologically active domain, which also includes a conserved DXD motif (Asp286/Asp288) and Trp102, which form a complex with Mn²⁺ and UDP-Glucose. The cysteine protease domain at residues 544-955 is necessary for autoproteolytic activity and delivery of the enzymatic domain into the cytosol. Between the cysteine protease domain and the C-terminal binding domain, a delivery domain is suggested, which is responsible for toxin translocation across membranes and delivers the glucosyltransferase into the cytosol of target cells. Finally, the fourth functional domain of TcdB is located within the carboxy-terminal region of the toxin (1851-2366), and is predicted to interact with receptors on target cells. However, the precise toxin receptor in humans has not been identified. After binding, the toxins are endocytosed via clathrin- and dynamin-dependent pathways to reach acidic endosomal compartments from where the toxins are translocated into the cytosol. Most likely in the cytosol, the cysteine protease domain is activated by binding of InsP6, resulting in autocleavage and release of the glucosyltransferase domain, which then targets Rho proteins (RhoA, -B, and -C), Rac and Cdc42. The glycosylation of these small regulatory proteins, lead to disruption of vital signaling pathways in the cells, resulting in actin condensation and consequent rounding of the cells, membrane blebbing, and eventual apoptosis and death of the target cell. While both TcdA and TcdB exert their activities on a wide range of cell types, TcdB exhibits a higher rate of enzymatic activity than TcdA, leading to a quickened rate of cytopathic effects in some cell types. Depending on the cell type, TcdB ranged from 4-fold to 200-fold more cytotoxic than TcdA in different studies.

There is an unmet need for effective treatment and/or prevention of C. difficile associated infections including prevention from relapse of CDAD. The present invention provides both mouse and humanized antibodies to toxin B to satisfy these and other needs.

SUMMARY

The invention comprises in one embodiment an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 110, 111, and 112, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 102, 103 and 104, respectively.

In another embodiment, the invention comprises an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 126, 127 and 128, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 118, 119 and 120, respectively.

In a third embodiment, the invention comprises an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 142, 143 and 144, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 134, 135 and 136, respectively.

In a fourth embodiment, the invention comprises an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 206, 207 and 208, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 198, 199 and 200, respectively.

In a fifth embodiment, the invention comprises an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 222, 223 and 224, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 214, 215 and 216, respectively.

In a sixth embodiment, the invention comprises an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain region and a light chain region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 238, 239 and 240, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 230, 231 and 232, respectively.

In a seventh embodiment, the invention comprises an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 238, 239 and 240, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 230, 231 and 232, respectively.

In an eighth embodiment the invention comprises an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain region, wherein the heavy chain variable region comprises amino acid sequences about 80% to about 100% homologous to SEQ ID NO. 710 and the light chain variable region comprises amino acid sequences about 80% to about 100% homologous to SEQ ID NO. 708.

In a ninth embodiment, the invention comprises an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 76, 77 and 78, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 68, 69 and 70, respectively. In this embodiment, the heavy chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 75, and wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 67.

In a tenth embodiment, the invention comprises an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 44, 45 and 46, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 36, 37 and 38, respectively. In this embodiment the heavy chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 43, and wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 35.

In an eleventh embodiment, the isolated monoclonal antibody or an antigen-binding portion thereof, that binds to C. difficile toxin B, comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 109, 125, 141, 157, 173, 189, 205, 221 and 237 and comprises a light chain variable region, wherein the light chain variable region having an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 101, 117, 133, 149, 165, 181, 197, 213 and 229.

In a twelfth embodiment, the isolated monoclonal antibody or an antigen-binding portion thereof, that binds to C. difficile toxin B, comprises a heavy chain variable region, wherein the heavy chain variable region comprises a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NOs: 389, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549, 565, 571, 587, 603, 619, 635, 651 and 709, and wherein the light chain variable region comprises a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NOs: 381, 397, 413, 429, 445, 461, 477, 493, 509, 525, 541, 557, 563, 579, 595, 611, 627, 643 and 707.

The present invention also provides for an isolated monoclonal antibody, or an antigen-binding portion, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 76, 77 and 78, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 68, 69 and 70, respectively.

The present invention provides for an isolated monoclonal antibody, or an antigen-binding portion, that binds to Clostridium difficile (C. difficile) toxin B and comprises: (1) a heavy chain variable region, wherein the heavy chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 76, 77 and 78, respectively; (2) a light chain variable region, wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 68, 69 and 70, respectively; (3) a heavy chain variable region, wherein the heavy chain comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 75, and wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 67; (4) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 44, 45 and 46, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 36, 37 and 38, respectively; (5) a heavy chain variable region, wherein the heavy chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 44, 45 and 46, respectively; (6) a light chain variable region, wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 36, 37 and 38, respectively; (7) a heavy chain variable region, wherein the heavy chain comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 43, and wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 35; (8) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 110, 111 and 112 respectively; (ii) SEQ ID NOs: 126, 127 and 128, respectively; or (iii) SEQ ID NOs: 142, 143 and 144, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 102, 103 and 104, respectively; (ii) SEQ ID NOs: 118, 119 and 120, respectively; or (iii) SEQ ID NOs: 134, 135 and 136, respectively; (9) a heavy chain variable region, wherein the heavy chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 110, 111 and 112 respectively; (ii) SEQ ID NOs: 126, 127 and 128, respectively; or (iii) SEQ ID NOs: 142, 143 and 144, respectively; (10) a light chain variable region, wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 102, 103 and 104 respectively; (ii) SEQ ID NOs: 118, 119 and 120, respectively; or (iii) SEQ ID NOs: 134, 135 and 136, respectively; (11) a heavy chain variable region, wherein the heavy chain comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 109, 125 or 141, and wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 101, 117 or 133; (12) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 206, 207 and 208, respectively; (ii) SEQ ID NOs: 222, 223 and 224, respectively; or (iii) SEQ ID NOs: 238, 239 and 240, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 198, 199 and 200 respectively; (ii) SEQ ID NOs: 214, 215 and 216, respectively; or (iii) SEQ ID NOs: 230, 231 and 232, respectively; (13) a heavy chain variable region, wherein the heavy chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 206, 207 and 208 respectively; (ii) SEQ ID NOs: 222, 223 and 224, respectively; or (iii) SEQ ID NOs: 238, 239 and 240, respectively; (14) a light chain variable region, wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 198, 199 and 200 respectively; (ii) SEQ ID NOs: 214, 215 and 216, respectively; or (iii) SEQ ID NOs: 230, 231 and 232, respectively; (15) a heavy chain variable region wherein the heavy chain comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 205, 221 or 237, and wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 197, 213 or 229; (16) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 486, 487 and 488, respectively; (ii) SEQ ID NOs: 502, 503 and 504, respectively; or (iii) SEQ ID NOs: 518, 519 and 520, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 478, 479 and 480, respectively; (ii) SEQ ID NOs: 494, 495 and 496, respectively; or (iii) SEQ ID NOs: 510, 511 and 512, respectively; (17) a heavy chain variable region, wherein the heavy chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 486, 487 and 488, respectively; (ii) SEQ ID NOs: 502, 503 and 504, respectively; or (iii) SEQ ID NOs: 518, 519 and 520, respectively; (18) a light chain variable region, wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 478, 479 and 480, respectively; (ii) SEQ ID NOs: 494, 495 and 496, respectively; or (iii) SEQ ID NOs: 510, 511 and 512, respectively; (19) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 390, 391 and 392, respectively; (ii) SEQ ID NOs: 406, 407 and 408, respectively; or (iii) SEQ ID NOs: 422, 423 and 424, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 382, 383 and 384, respectively; (ii) SEQ ID NOs: 398, 399 and 400, respectively; or (iii) SEQ ID NOs: 414, 415 and 416, respectively; (20) a heavy chain variable region, wherein the heavy chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 390, 391 and 392, respectively; (ii) SEQ ID NOs: 406, 407 and 408, respectively; or (iii) SEQ ID NOs: 422, 423 and 424, respectively; (21) a light chain variable region, wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 382, 383 and 384, respectively; (ii) SEQ ID NOs: 398, 399 and 400, respectively; or (iii) SEQ ID NOs: 414, 415 and 416, respectively; (22) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 620, 621 and 622 respectively; (ii) SEQ ID NOs: 636, 637 and 638, respectively; or (iii) SEQ ID NOs: 652, 653 and 654, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 612, 613 and 614, respectively; (ii) SEQ ID NOs: 628, 629 and 630, respectively; or (iii) SEQ ID NOs: 644, 645 and 646, respectively; (23) a heavy chain variable region, wherein the heavy chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 620, 621 and 622 respectively; (ii) SEQ ID NOs: 636, 637 and 638, respectively; or (iii) SEQ ID NOs: 652, 653 and 654, respectively; (24) a light chain variable region, wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 612, 613 and 614, respectively; (ii) SEQ ID NOs: 628, 629 and 630, respectively; or (iii) SEQ ID NOs: 644, 645 and 646, respectively; (25) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 534, 535 and 536, respectively; (ii) SEQ ID NOs: 550, 551 and 552, respectively; or (iii) SEQ ID NOs: 566, 567 and 568, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 526, 527 and 528, respectively; (ii) SEQ ID NOs: 542, 543 and 544, respectively; or (iii) SEQ ID NOs: 558, 559 and 560, respectively; (26) a heavy chain variable region, wherein the heavy chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 534, 535 and 536, respectively; (ii) SEQ ID NOs: 550, 551 and 552, respectively; or (iii) SEQ ID NOs: 566, 567 and 568, respectively; (27) a light chain variable region, wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 526, 527 and 528, respectively; (ii) SEQ ID NOs: 542, 543 and 544, respectively; or (iii) SEQ ID NOs: 558, 559 and 560, respectively; (28) a heavy chain variable region encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NO: 485, 501 and 517 wherein the light chain variable region is encoded by an nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NO: 477, 493 and 509; (29) a heavy chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NO: 389, 405 or 421, and wherein the light chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NO: 381, 397 or 413; (30) a heavy chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NO: 619, 635 or 651, and wherein the light chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NO: 611, 627 or 643; (31) a heavy chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NO: 533, 549 or 565, and wherein the light chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NO: 525, 541 or 557; (32) a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 11, 27, 43, 59, 75 or 93; (33) a light chain variable region, wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 3, 19, 35, 51, 67 or 85; (34) a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 109, 125, 141, 157, 173, 189, 205, 221, 237 or 710; (35) a light chain variable region, wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 101, 117, 133, 149, 165, 181, 197, 213, 229 or 708; (36) a heavy chain variable region, wherein the heavy chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NOs: 253, 269, 285, 301, 317, 341, 357 or 373; (37) a light chain variable region, wherein the light chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NOs: 245, 261, 277, 293, 309, 325, 333, 349 or 365; (38) a heavy chain variable region, wherein the heavy chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NOs: 389, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549, 565, 571, 587, 603, 619, 635 or 651; (39) a light chain variable region, wherein the light chain variable region is encoded by a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NOs: 381, 397, 413, 429, 445, 461, 477, 493, 509, 525, 541, 557, 579, 595, 611, 627, 643 or 714; or (40) a heavy chain variable region and a light chain variable region comprising amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 75 and 67, respectively.

The isolated monoclonal antibody or antigen-binding portion thereof binds to C. difficile toxin B, and may have a dissociation constant (K_(D)) less than about 1×10⁻⁸ M.

The isolated monoclonal antibody or antigen-binding portion may be humanized or chimeric, e.g., mouse-human, and may be: (a) a whole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab′)₂; or (e) a disulfide linked Fv and may contain at least one constant domain, e.g., (a) an IgG constant domain; (b) IgM constant domain; (c) IgD constant domain; (d) IgE constant domain; or (e) an IgA constant domain. The present antibody or antigen-binding portion may be fused in-frame to fusion partners or incorporate domains for post-translational modifications to facilitate stability in vitro to affect formulation/shelf life (e.g. encapsulation, acylated) or in vivo to affect PK/PD (e.g. pegylation, sialyation, glycosylation). The fusion partner may act as a ligand for therapeutic or diagnostic applications. The antibody or antigen-binding portion may be engineered to increase avidity through valency (multivalent) or complimented with specificity, by fusion or association with antibody or antigen-binding portions against other antigens (e.g. different domains of TcdA, TcdB, binary toxin).

The isolated monoclonal antibody or antigen-binding portion may bind to: (i) fragment 4 of C. difficile toxin B; and/or, (ii) fragment 1 of C. difficile toxin B.

The present invention provides for an isolated monoclonal antibody, or an antigen-binding portion, wherein the antibody, or antigen-binding portion thereof, binds to the same or antigenically similar epitope of C. difficile toxin B recognized by an antibody comprising: (1) a heavy chain variable region and a light chain variable region comprising amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 43 and 35, respectively; (2) a heavy chain variable region and a light chain variable region comprising amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 109 and 101, respectively; (ii) SEQ ID NOs: 125 and 117, respectively; (iii) SEQ ID NOs: 141 and 133, respectively; (3) a heavy chain variable region and a light chain variable region comprising amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in (i) SEQ ID NOs: 205 and 197, respectively; (ii) SEQ ID NOs: 221 and 213, respectively; or (iii) SEQ ID NOs: 237 and 229, respectively; (4) a heavy chain variable region and a light chain variable region comprising nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 389 and 381, respectively; (ii) SEQ ID NOs: 405 and 397, respectively; (iii) SEQ ID NOs: 421 and 413, respectively; or (iv) SEQ ID NOs: 709 and 707, respectively; or, (5) a heavy chain variable region and a light chain variable region encoded by nucleic acid sequences about 80% to about 100% homologous to the nucleic acid sequences set forth in (i) SEQ ID NOs: 485 and 477, respectively; (ii) SEQ ID NOs: 501 and 493, respectively; or (iii) SEQ ID NOs: 517 and 509, respectively.

The present invention provides for an isolated monoclonal antibody produced by hybridoma designated CAN33G1, CAN46G4, CAN46G13, CAN46G13a, CAN46G19 or CAN46G24 or for a hybridoma which is designated CAN33G1, CAN46G4, CAN46G13, CAN46G13a, CAN46G19 or CAN46G24.

The present invention provides for an isolated monoclonal antibody, or an antigen-binding portion thereof, wherein, in an in vivo toxin B challenge experiment, when the antibody, or an antigen-binding portion thereof, is administered to a mammal at a dosage ranging from about 8 mg/kg body weight to about 13 mg/kg body weight about 24 hours before the mammal is exposed to about 75 ng or greater than about 75 ng of C. difficile toxin B, the chance of survival for the mammal is greater than about 80% within about 4 days after the exposure to toxin B. Lethal dose or lethal concentration is dependent on the toxicity of the toxin. The amount of antibody required to neutralize the toxin may vary accordingly.

The present invention provides for an isolated monoclonal antibody, or an antigen-binding portion thereof, wherein the antibody, or antigen-binding portion thereof, at a concentration ranging from about 25 μg/ml to about 100 μg/ml, neutralizes greater than about 40% of about 5 ng/ml C. difficile toxin B in an in vitro neutralization assay. The toxicity of the toxin is dependent on the strain from which it was isolated, as it varies between strains. Accordingly, the concentration of antibody required to neutralize the toxin is dependent on the source of the toxin.

Cells that may be used with the present invention, include, but are not limited to, bacterial cell, a eukaryotic cell, or a mammalian cell. For example, the cells can be COS-1, COS-7, HEK293, BHK21, CHO, CHOK1SV, Per.C6, BSC-1, Hep G2, SP2/0, HeLa, myeloma or lymphoma cells.

The present invention provides for an antibody produced by a hybridoma designated: (1) CAN46G13-1-8, wherein the hybridoma is deposited with the American Type Culture Collection having the ATCC Patent Deposit Designation PTA-13257. The deposit for PTA-13257 was made on Aug. 23, 2012. As used herein, CAN46G13a refers to the hybridoma clone CAN46G13-1-8 or the monoclonal antibodies generated by the corresponding clone; (2) CAN46G4-1-2, wherein the hybridoma is deposited with the American Type Culture Collection having the ATCC Patent Deposit Designation PTA-13258. The deposit for PTA-13258 was made on Aug. 23, 2012. As used herein, CAN46G4 refers to the clone CAN46G4-1-2 or the monoclonal antibodies generated by the corresponding clone; (3) CAN46G19-3-2, wherein the hybridoma is deposited with the American Type Culture Collection having the ATCC Patent Deposit Designation PTA-13259. The deposit for PTA-13259 was made on Aug. 23, 2012. As used herein, CAN46G19 refers to the clone CAN46G19-3-2 or the monoclonal antibodies generated by the corresponding clone; (4) CAN46G13-1-5, wherein the hybridoma is deposited with the American Type Culture Collection having the ATCC Patent Deposit Designation PTA-13260. The deposit for PTA-13260 was made on Aug. 23, 2012. As used herein, CAN46G13 and CAN46G24 refer to the clone CAN46G13-1-5 or the monoclonal antibodies generated by the corresponding clone. The sequences for the monoclonal antibodies generated by CAN46G13 and CAN46G24 are identical.

The present invention provides for a composition comprising the isolated monoclonal antibody or antigen-binding portion thereof, and at least one pharmaceutically acceptable carrier. This composition may be used as a method of preventing or treating C. difficile-associated disease comprising administering to a subject an effective amount of the present antibody or antigen-binding portion thereof. The antibody or antigen-binding portion thereof may be administered intravenously, subcutaneously, intramuscularly or transdermally. The present method may further comprise the step of administering to the subject a second agent, or multiple agents (e.g., third, fourth, fifth and sixth) such as a different antibody or fragment thereof (e.g., an antibody or antigen-binding portion thereof that binds C. difficile toxin A), an antiparasitic (e.g. nitrazoxanide), an antibiotic (e.g., vancomycin, metronidazole, rifaximin, or fidaxomicin), probiotics (compositions with saccharomyces boulardi, bifidobacteria, or lactobacillus), or fecal transplant. The present method may further comprise the step of administering to the subject one or more additional agents such as a different antibody or fragment thereof (e.g., and antibody or antigen-bind portion thereof that binds a different fragment of C. difficile toxin B).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an ELISA showing the binding specificity of murine CAN33 and CAN46 monoclonal antibodies (mAbs) to whole toxin B (TcdB), fragment 4 of toxin B (TcdB F4), and fragment 1 of toxin B (TcdB F1). TcdA (toxin A) negative control is shown, along with control antibodies hPA-41, mouse anti-toxin B polyclonal (mouse pAB), and anti-toxin B (CAN20G2).

FIG. 2 is a competition ELISA showing the murine CAN33 and CAN46 mAbs bind distinct epitopes and do not compete with mAb MDX-1388 (Medarex) on toxin B.

FIG. 3 is a competition ELISA showing the murine CAN33 and CAN46 mAbs bind distinct epitopes and do not compete with mAb hPA-41 (Progenics Pharmaceuticals, Inc.) on toxin B.

FIG. 4 shows a Western immunoblot of purified murine CAN46G4 and CAN46G19 mAbs.

FIG. 5 shows a Western immunoblot of purified murine CAN46G13 and CAN46G13a mAbs.

FIG. 6 shows a Western immunoblot of purified murine CAN46G24 mAb.

FIG. 7 is an epitope binning graph for murine CAN46G4, CAN46G13a and CAN46G24.

FIG. 8 shows a 4-PL toxin titration curve of TcdB on HT-29 cells using the xCelligence platform.

FIG. 9 is a bar graph showing the effects of C. difficile toxin B on mouse survival and the efficacy of the murine CAN33 and murine CAN46 mAbs against the toxin B challenge.

FIG. 10 shows primers used for variable (V) chain gene amplification from RNA.

FIG. 11 shows variable (V) gene sequencing results for murine CAN46G4, CAN46G13a, CAN46G19, CAN46G24, CAN46G13, and CAN33G1 that includes, both VH and VL sequences from the murine CAN46 and CAN33G1 parental clones.

FIG. 12 shows amino acid variable V-region sequence of humanized CDR Grafted CAN46 mAbs.

FIG. 13 shows amino acid variable V-region sequence of humanized huCAN46G mAbs.

FIG. 14 shows amino acid variable V-region sequence of resurfaced, humanized rehuCAN46G mAbs.

FIG. 15 a shows a bar graph depicting in vitro neutralization data for purified humanized CAN46G4 variants in Per.C6 construct expressed in HEK293F cells at 250 pg/ml depicted as a bar graph.

FIG. 15 b shows a bar graph depicting in vitro neutralization data for purified humanized CAN46G19 variants in Per.C6 construct expressed in HEK293F cells at 250 pg/ml depicted as a bar graph.

FIG. 16 is a Kaplan-Meier plot showing the effects of C. difficile toxin B on mouse survival and the efficacy of the humanized CAN46G13a mAbs (purified from HEK293F cells expressing the Per.C6-based construct) against the toxin B challenge.

FIG. 17 is a Kaplan-Meier plot showing the effects of C. difficile toxin B on mouse survival and the efficacy of the humanized CAN46 mAbs (purified from HEK293F cells expressing the Per.C6-based construct) against the toxin B challenge.

FIG. 18 is a table showing the total Human IgG ELISA results from mice injected with humanized CAN46 mAb pre- (−12 hours) and post- (72 hours after challenge) Toxin B challenge.

FIG. 19 is a line graph showing the in vitro Toxin B neutralization of the humanized CAN46G24 mAbs

FIG. 20 is a line graph showing the in vitro Toxin B neutralization of the humanized CAN46G13a mAbs

FIG. 21 is a line graph showing the in vitro Toxin B neutralization of the humanized CAN46G19 mAbs.

FIG. 222 is a line graph showing the in vitro Toxin B neutralization of the humanized huCAN46G mAbs.

FIG. 23 is a line graph showing the in vitro Toxin B neutralization capabilities of the humanized CAN46G mAbs.

FIG. 24 is a table showing the affinity analysis of humanized CAN46G4, CAN46G13a, and CAN46G19 Tcd B mAbs.

FIG. 25 is a line graph showing the in vitro Toxin B neutralization of the humanized CAN46G mAbs purified from CHOK1 SV cells expressing the CHO-based construct

FIG. 26 includes multiple bar graphs showing the EC50 of the humanized CAN46 mAbs against various C. difficile clinical isolates.

FIG. 29 is a Kaplan-Meier plot showing the protective effects on hamster survival of human CDA1/MDX1388 and humanized HeCAN20G2/HuCAN46G24 mAbs doses against infection with B1 C. difficile spores

FIG. 28A shows the change from baseline in body weight of the hamsters after infection with C. difficile B1 spores.

FIG. 28B shows the level of toxin specific human CDA1/MDX1388 mAbs and humanized HeCAN20G2/HuCAN46G24 mAbs in hamsters infected with C. difficile B1 spores.

FIG. 29 is a table showing the immunoreactive responses in vitro measured by direct ELISA of different mAbs purified from HEK293F cells expressing the Per.C6-based constructs against partially purified toxins from different C. difficile strains.

FIG. 30 shows the % Neutralization of different antibody combinations as determined from independent assays conducted across different experiments for the B1 and NAP1 strains (BI-1, BI-6, and BI-17)

FIG. 31 contains graphs showing the binding characteristics of humanized CAN46 mAbs to captured toxins from different C. difficile non-NAP1 and NAP1 strains by sandwich ELISA.

FIG. 32 is a graph showing the immunoreactivity of humanized CAN46 to captured toxins from different C. difficile non-NAP1 strains by ELISA.

FIG. 33 is a graph showing the immunoreactivity of humanized CAN46G mAbs to captured toxin B from different NAP1 C. difficile strains by ELISA.

FIG. 34 shows Western immunoblots of humanized CAN46 mAbs purified from CHOKS1V cells expressing the CHO construct.

FIG. 35 shows Western immunoblots of humanized CAN46 mAbs purified from HEK293 cells expressing the Per.C6-based construct.

FIG. 36 is a table showing the affinity analysis of purified Tcd B humanized mAbs CAN46G24 and CAN46G13a from CHOK1SV cells expressing the CHO-based constructs.

FIG. 37 is a bar graph showing the binding specificities of humanized CAN46 mAbs against Toxin B and Toxin A.

DETAILED DESCRIPTION

The present invention provides for compositions and methods for the diagnosis, prevention or treatment of Clostridium difficile (C. difficile) bacterial infection or bacterial carriage. The compositions contain antibodies (or an antigen-binding portion) that recognize toxin B of C. difficile, including mouse monoclonal antibodies, humanized antibodies, chimeric antibodies (murine/human), or antigen-binding portions of any of the foregoing. These antibodies (or antigen-binding portion thereof) can neutralize toxin B in vitro, in vivo, and/or inhibit binding of toxin B to mammalian cells. Therefore, the present antibodies or antigen-binding portion can be used in a passive immunization manner or protocol to prevent or treat C. difficile-associated disease (CDAD).

In one embodiment, the present antibodies or antigen-binding portion provide one or more of the following effects: protect from or treat C. difficile-mediated colitis, antibiotic-associated colitis, pseudomembranous colitis (PMC) or other intestinal disease in a subject; protect from or treat diarrhea in a subject; and/or treat or inhibit relapse of C. difficile-mediated disease. When administered to a mammal, the present antibodies or antigen-binding portion may protect the mammal against toxin B administered in an amount that would otherwise be fatal to the mammal had the antibody or antigen-binding portion not administered.

The present antibodies or antigen-binding portions include murine antibodies produced by hybridomas CAN46G4, CAN46G13, CAN46G13a, CAN46G19, CAN46G24 and CAN33G1 as well as humanized antibodies derived from the same hybridomas described herein.

Also encompassed by the present invention are antibodies or antigen-binding portions that include an antigen-binding portion of the antibody produced by hybridomas CAN46G4, CAN46G13, CAN46G13a, CAN46G19, CAN46G24 or CAN33G1; as used herein, CAN46G4, CAN46G13, CAN46G13a, CAN46G19, CAN46G24 and CAN33G1 refer to the hybridoma clones or the monoclonal antibodies generated by the corresponding hybridoma clones.

The antibodies or antigen-binding portions can specifically bind to an epitope: (i) within fragment 1 of toxin B, e.g., an epitope between amino acid residues 1-592 of toxin B (CAN46G13a); or an epitope within fragment 4 of toxin B, e.g., an epitope between amino acid residues 1777-2366 of toxin B (CAN46G4, CAN46G13, CAN46G19, CAN46G24 or CAN33G1). Babcock, G. J. et al., Infection and Immunity, 74: 6339-6347 (2006).

In other embodiments, the antibodies or antigen-binding portions specifically bind to an epitope within fragment 2 (amino acid residues 593-1183) or fragment 3 (amino acid residues 1184-1776) of toxin B. In certain embodiments, the antibodies or antigen-binding portions specifically bind an epitope within amino acid residues 1-600, 400-600, 415-540, 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 900-1000, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1800-1900, 1900-2000, 2000-2100, 2100-2200 or 2200-2366 of toxin B, or any interval, portion or range thereof.

The present antibodies, or antigen-binding portions, include, but are not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, polyclonal antibodies, recombinant antibodies, as well as antigen-binding portions of the foregoing. An antigen-binding portion of an antibody may include a portion of an antibody that specifically binds to a toxin of C. difficile (e.g., toxin B) and may comprise the heavy or light chain alone of the antibody molecule.

CDRs and Variable Regions

The CDRs of the present antibodies or antigen-binding portions can be from a non-human, e.g., murine (Mus musculus) or a human source (Homo Saipian). The framework of the present antibodies or antigen-binding portions can be human, humanized, non-human (e.g., a murine framework modified to decrease antigenicity in humans), or a synthetic framework (e.g., a consensus sequence).

In one embodiment, the present antibodies, or antigen-binding portions, contain at least one heavy chain variable region and/or at least one light chain variable region. The heavy chain variable region (or light chain variable region) contains three CDRs and four framework regions (FRs), arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Kabat, E. A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991. Chothia, C. et al., J. Mol. Biol. 196:901-917, 1987.

The present antibodies or antigen-binding portions can specifically bind to toxin B with a dissociation constant (K_(D)) of less than about 10⁻⁷ M, less than about 10⁻⁸ M, less than about 10⁻⁹ M, less than about 10⁻¹⁰ M, less than about 10⁻¹¹ M, or less than about 10⁻¹² M.

Antibodies with a heavy chain variable region and a light chain variable region that are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the heavy chain variable region and light chain variable region of the antibody produced by clone CAN46G4, CAN46G13, CAN46G13a, CAN46G19, CAN46G24 or CAN33G1 can also bind to toxin B and are encompassed by the invention.

In related embodiments, anti-toxin B antibodies or antigen-binding portions include, for example, the CDRs of variable heavy chains and/or variable light chains of CAN46G4, CAN46G13, CAN46G13a, CAN46G19, CAN46G24 or CAN33G1. The CDRs of the heavy chain variable regions from these clones, as well as the CDRs of the light chain variable regions from these clones, are shown in Table 1.

TABLE 1 Sequence ID NOs Seq Chain, ID Name Region Origin Sequence No: Fragment 1  TcdB, Clostridium MSLVNRKQLEKMANVRFRTQEDEYV   1 of Toxin B Frag 1, difficile AILDALEEYHNMSENTVVEKYLKLKDI aa 1- NSLTDIYIDTYKKSGRNKALKKFKEYL 546 VTEVLELKNNNLTPVEKNLHFVWIGG QINDTAINYINQWKDVNSDYNVNVFY DSNAFLINTLKKTVVESAINDTLESFRE NLNDPRFDYNKFFRKRMEIIYDKQKNF INYYKAQREENPELIIDDIVKTYLSNEY SKEIDELNTYIEESLNKITQNSGNDVRN FEEFKNGESFNLYEQELVERWNLAAAS DILRISALKEIGGMYLDVDMLPGIQPDL FESIEKPSSVTVDFWEMTKLEAIMKYK EYIPEYTSEHFDMLDEEVQSSFESVLAS KSDKSEIFSSLGDMEASPLEVKIAFNSK GIINQGLISVKDSYCSNLIVKQIENRYKI LNNSLNPAISEDNDFNTTTNTFIDSIMA EANADNGRFMMELGKYLRVGFFPDV KTTINLSGPEAYAAAYQDLLMFKEGS MNIHLIEADLRNFEISKTNISQSTEQEM ASLWSFDDARAKAQFEEYKRNYFEGS LGED Fragment 4  TcdB, Clostridium ANKLSFNFSDKQDVPVSEIILSFTPSYY   2 of Toxin B Frag 4, difficile EDGLIGYDLGLVSLYNEKFYINNFGM aa MVSGLIYINDSLYYFKPPVNNLITGFVT 1777- VGDDKYYFNPINGGAASIGETIIDDKN 2366 YYFNQSGVLQTGVFSTEDGFKYFAPA NTLDENLEGEAIDFTGKLIIDENIYYFD DNYRGAVEWKELDGEMHYFSPETGK AFKGLNQIGDYKYYFNSDGVMQKGFV SINDNKHYFDDSGVMKVGYTEIDGKH FYFAENGEMQIGVFNTEDGFKYFAHH NEDLGNEEGEEISYSGILNFNNKIYYFD DSFTAVVGWKDLEDGSKYYFDEDTAE AYIGLSLINDGQYYFNDDGIMQVGFVT INDKVFYFSDSGIIESGVQNIDDNYFYI DDNGIVQIGVFDTSDGYKYFAPANTVN DNIYGQAVEYSGLVRVGEDVYYFGET YTIETGWIYDMENESDKYYFNPETKKA CKGINLIDDIKYYFDEKGIMRTGLISFE NNNYYFNENGEMQFGYINIEDKMFYF GEDGVMQIGVFNTPDGFKYFAHQNTL DENFEGESINYTGWLDLDEKRYYFTDE YIAATGSVIIDGEEYYFDPDTAQLVISE CAN46G4 K, Mus EKVLTQSPAIMSASPGEEVTMTCSASSS   3 variable musculus VSYMHWYQQKSSTSPKLWIYETSKLA region FGVPGRFSGSGSGNSYSLTISSMEAEDV ATYYCFQGSGYPFTFGSGTKLEVK CAN46G4 K, Mus SSVSY   4 CDR1 musculus CAN46G4 K, Mus ETS   5 CDR2 musculus CAN46G4 K, Mus FQGSGYPFT   6 CDR3 musculus CAN46G4 K, Mus EKVLTQSPAIMSASPGEEVTMTCSAS   7 FR1 musculus CAN46G4 K, Mus MHWYQQKSSTSPKLWIY   8 FR2 musculus CAN46G4 K, Mus KLAFGVPGRFSGSGSGNSYSLTISSMEA   9 FR3 musculus EDVATYYC CAN46G4 K, Mus FGSGTKLEVK  10 FR4 musculus CAN46G4 H, Mus EVQLLQSGPELVKPGASVKISCKASDY  11 variable musculus SFTGYYMHWVKQSHVKSLEWIGRIFP region YNGAASYNQNFKDKATLTVDKSSSTA YMELHSLTSEDSAVYYCTRWLRVYFD YWGQGTTLTVSS CAN46G4 H, Mus DYSFTGYY  12 CDR1 musculus CAN46G4 H, Mus IFPYNGAA  13 CDR2 musculus CAN46G4 H, Mus TRWLRVYFDY  14 CDR3 musculus CAN46G4 H, Mus EVQLLQSGPELVKPGASVKISCKAS  15 FR1 musculus CAN46G4 H, Mus MHWVKQSHVKSLEWIGR  16 FR2 musculus CAN46G4 H, Mus SYNQNFKDKATLTVDKSSSTAYMELH  17 FR3 musculus SLTSEDSAVYYC CAN46G4 H, Mus WGQGTTLTVSS  18 FR4 musculus CAN46G13 K, Mus EIVLTQSPAIMSTSPGEKVTMSCSASSS  19 variable musculus VTYMHWYQQKSITSPKLWIYETSKLAS region GVPGRFSGSGSGNSYSLTISSMEAEDV ATYYCFQGSGYPFTFGSGTKLEIK CAN46G13 K, Mus SSVTY  20 CDR1 musculus CAN46G13 K, Mus ETS  21 CDR2 musculus CAN46G13 K, Mus FQGSGYPFT  22 CDR3 musculus CAN46G13 K, Mus EIVLTQSPAIMSTSPGEKVTMSCSAS  23 FR1 musculus CAN46G13 K, Mus MHWYQQKSITSPKLWIY  24 FR2 musculus CAN46G13 K, Mus KLASGVPGRFSGSGSGNSYSLTISSMEA  25 FR3 musculus EDVATYYC CAN46G13 K, Mus FGSGTKLEIK  26 FR4 musculus CAN46G13 H, Mus EVQLLQSGPELVKPGTSVKISCKASGY  27 variable musculus SFTGYYIHWVKQTHVKSLEWVGRIFPY region NGAASYNQNFKGKATLTVDKSSSTAY MELHSLTSEDSAVYFCARWLRVYFDY WGQGTTLTVSS CAN46G13 H, Mus GYSFTGYY  28 CDR1 musculus CAN46G13 H, Mus IFPYNGAA  29 CDR2 musculus CAN46G13 H, Mus ARWLRVYFDY  30 CDR3 musculus CAN46G13 H, Mus EVQLLQSGPELVKPGTSVKISCKAS  31 FR1 musculus CAN46G13 H, Mus IHWVKQTHVKSLEWVGR  32 FR2 musculus CAN46G13 H, Mus SYNQNFKGKATLTVDKSSSTAYMELH  33 FR3 musculus SLTSEDSAVYFC CAN46G13 H, Mus WGQGTTLTVSS  34 FR4 musculus CAN46G13a K, Mus ENVLTQSPAIMAASLGQKVTMTCSASS  35 variable musculus SVSSSYLHWYQQKSGASPKPLIHRTST region LASGVPARFSGSGSGTSYSLTISSVEAE DDATYYCQQWSGYPYTFGGGTKLEIK CAN46G13a K, Mus SSVSSSY  36 CDR1 musculus CAN46G13a K, Mus RTS  37 CDR2 musculus CAN46G13a K, Mus QQWSGYPYT  38 CDR3 musculus CAN46G13a K, Mus ENVLTQSPAIMAASLGQKVTMTCSAS  39 FR1 musculus CAN46G13a K, Mus LHWYQQKSGASPKPLIH  40 FR2 musculus CAN46G13a K, Mus TLASGVPARFSGSGSGTSYSLTISSVEA  41 FR3 musculus EDDATYYC CAN46G13a K, Mus FGGGTKLEIK  42 FR4 musculus CAN46G13a H, Mus DVQLQESGPGLVKPSQSLSLTCTVTGY  43 variable musculus SITSDSAWNWIRQFPGNNLEWMGYISY region SGSTSYNPSLKSRISITRDTSKNQFFLQL NSVTTEDTATYYCARRSRVSFYFDYW GQGTTLTVSS CAN46G13a H, Mus GYSITSDSA  44 CDR1 musculus CAN46G13a H, Mus ISYSGST  45 CDR2 musculus CAN46G13a H, Mus ARRSRVSFYFDY  46 CDR3 musculus CAN46G13a H, Mus DVQLQESGPGLVKPSQSLSLTCTVT  47 FR1 musculus CAN46G13a H, Mus WNWIRQFPGNNLEWMGY  48 FR2 musculus CAN46G13a H, Mus SYNPSLKSRISITRDTSKNQFFLQLNSV  49 FR3 musculus TTEDTATYYC CAN46G13a H, Mus WGQGTTLTVSS  50 FR4 musculus CAN46G19 K, Mus ENVLTQSPTIMSASPGEEVTMTCSASSS  51 variable musculus VTYMHWYQQKSITSPKLWIYETSKLAS region GVPGRFSGSGSGNSYSLTISSMEAEDV ATYYCFQGSGYPFTFGSGTKLEIK CAN46G19 K, Mus SSVTY  52 CDR1 musculus CAN46G19 K, Mus ETS  53 CDR2 musculus CAN46G19 K, Mus FQGSGYPFT  54 CDR3 musculus CAN46G19 K, Mus ENVLTQSPTIMSASPGEEVTMTCSAS  55 FR1 musculus CAN46G19 K, Mus MHWYQQKSITSPKLWIY  56 FR2 musculus CAN46G19 K, Mus KLASGVPGRFSGSGSGNSYSLTISSMEA  57 FR3 musculus EDVATYYC CAN46G19 K, Mus FGSGTKLEIK  58 FR4 musculus CAN46G19 H, Mus EVQLLQSGPELVKPGTSVKISCKASGY  59 variable musculus SFTGYYIHWVKQTHVKSLEWVGRIFPY region NGAASYNQNFKGKATLTVDKSSTTAY MELHSLTSEDSAVYFCARWLRVYFDY WGQGTTLTVSS CAN46G19 H, Mus GYSFTGYY  60 CDR1 musculus CAN46G19 H, Mus IFPYNGAA  61 CDR2 musculus CAN46G19 H, Mus ARWLRVYFDY  62 CDR3 musculus CAN46G19 H, Mus EVQLLQSGPELVKPGTSVKISCKAS  63 FR1 musculus CAN46G19 H, Mus IHWVKQTHVKSLEWVGR  64 FR2 musculus CAN46G19 H, Mus SYNQNFKGKATLTVDKSSTTAYMELH  65 FR3 musculus SLTSEDSAVYFC CAN46G19 H, Mus WGQGTTLTVSS  66 FR4 musculus CAN46G24 K, Mus EIVLTQSPAIMSTSPGEKVTMSCSASSS  67 variable musculus VTYMHWYQQKSITSPKLWIYETSKLAS region GVPGRFSGSGSGNSYSLTISSMEAEDV ATYYCFQGSGYPFTFGSGTKLEIK CAN46G24 K, Mus SSVTY  68 CDR1 musculus CAN46G24 K, Mus ETS  69 CDR2 musculus CAN46G24 K, Mus FQGSGYPFT  70 CDR3 musculus CAN46G24 K, Mus EIVLTQSPAIMSTSPGEKVTMSCSAS  71 FR1 musculus CAN46G24 K, Mus MHWYQQKSITSPKLWIY  72 FR2 musculus CAN46G24 K, Mus KLASGVPGRFSGSGSGNSYSLTISSMEA  73 FR3 musculus EDVATYYC CAN46G24 K, Mus FGSGTKLEIK  74 FR4 musculus CAN46G24 H, Mus EVQLLQSGPELVKPGTSVKISCKASGY  75 variable musculus SFTGYYIHWVKQTHVKSLEWVGRIFPY region NGAASYNQNFKGKATLTVDKSSSTAY MELHSLTSEDSAVYFCARWLRVYFDY WGQGTTLTVSS CAN46G24 H, Mus GYSFTGYY  76 CDR1 musculus CAN46G24 H, Mus IFPYNGAA  77 CDR2 musculus CAN46G24 H, Mus ARWLRVYFDY  78 CDR3 musculus CAN46G24 H, Mus EVQLLQSGPELVKPGTSVKISCKAS  79 FR1 musculus CAN46G24 H, Mus IHWVKQTHVKSLEWVGR  80 FR2 musculus CAN46G24 H, Mus SYNQNFKGKATLTVDKSSSTAYMELH  81 FR3 musculus SLTSEDSAVYFC CAN46G24 H, Mus WGQGTTLTVSS  82 FR4 musculus CAN33G1 K, Mus DIQLTQSSSSFSVSLGDRVTITCKASEDI  85 variable musculus YNRLAWYQQRPGNAPRLLISGATSLET region GIPSRFSGSGSGKEYTLSIASLQTEDFVT YYCQQYWNIPTFGGGTRLEIK CAN33G1 K, Mus EDIYNR  86 CDR1 musculus CAN33G1 K, Mus GAT  87 CDR2 musculus CAN33G1 K, Mus QQYWNIPT  88 CDR3 musculus CAN33G1 K, Mus DIQLTQSSSSFSVSLGDRVTITCKAS  89 FR1 musculus CAN33G1 K, Mus LAWYQQRPGNAPRLLIS  90 FR2 musculus CAN33G1 K, Mus SLETGIPSRFSGSGSGKEYTLSIASLQTE  91 FR3 musculus DFVTYYC CAN33G1 K, Mus FGGGTRLEIK  92 FR4 musculus CAN33G1 H, Mus EVQLQQSGPDLVKPGASVKISCKASGY  93 variable musculus SFTGYYMHWVKQSHGKSLEWIGRVNP region YNGDTNYNQNFKDKAILTVDKSASTA YMEFRSLTSEDSAVYYCTRSNWENYF DYWGQGSTLTVSS CAN33G1 H, Mus GYSFTGYY  94 CDR1 musculus CAN33G1 H, Mus VNPYNGDT  95 CDR2 musculus CAN33G1 H, Mus TRSNWENYFDY  96 CDR3 musculus CAN33G1 H, Mus EVQLQQSGPDLVKPGASVKISCKAS  97 FR1 musculus CAN33G1 H, Mus MHWVKQSHGKSLEWIGR  98 FR2 musculus CAN33G1 H, Mus NYNQNFKDKAILTVDKSASTAYMEFR  99 FR3 musculus SLTSEDSAVYYC CAN33G1 H, Mus WGQGSTLTVSS 100 FR4 musculus cdrCAN46G4 K, Artificial EIVLTQSPATLSLSPGERATLSCSASSSV 737 variable sequence SYMHWYQQKPGQAPRLLIYETSKLAF region GIPARFSGSGSGTDFTLTISSLEPEDFAV YYCFQGSGYPFTFGQGTRLEIK cdrCAN46G4 K, Artificial SSVSY 738 CDR1 sequence cdrCAN46G4 K, Artificial ETS 739 CDR2 sequence cdrCAN46G4 K, Artificial FQGSGYPFT 740 CDR3 sequence cdrCAN46G4 K, Artificial EIVLTQSPATLSLSPGERATLSCSAS 741 FR1 sequence cdrCAN46G4 K, Artificial MHWYQQKPGQAPRLLIY 742 FR2 sequence cdrCAN46G4 K, Artificial KLAFGIPARFSGSGSGTDFTLTISSLEPE 743 FR3 sequence DFAVYYC cdrCAN46G4 K, Artificial FGQGTRLEIK 744 FR4 sequence cdrCAN46G4 H, Artificial QVQLVQSGAEVKKPGSSVKVSCKASG 745 variable sequence YTFTGYYMHWVRQAPGQGLEWIGRIF region PYNGAASYNQNFKDKATITADESTNT AYMELSSLRSEDTAVYYCARWLRVYF DYWGQGTLVTVSS cdrCAN46G4 H, Artificial GYTFTGYY 746 CDR1 sequence cdrCAN46G4 H, Artificial IFPYNGAA 747 CDR2 sequence cdrCAN46G4 H, Artificial ARWLRVYFDY 748 CDR3 sequence cdrCAN46G4 H, Artificial QVQLVQSGAEVKKPGSSVKVSCKAS 749 FR1 sequence cdrCAN46G4 H, Artificial MHWVRQAPGQGLEWIGR 750 FR2 sequence cdrCAN46G4 H, Artificial SYNQNFKDKATITADESTNTAYMELSS 751 FR3 sequence LRSEDTAVYYC cdrCAN46G4 H, Artificial WGQGTLVTVSS 752 FR4 sequence huCAN46G4 K, Artificial EKVLTQSPATLSLSPGERATMTCSASSS 753 variable sequence VSYMHWYQQKPGTSPKLWIYETSKLA region FGVPARFSGSGSGNSYSLTISSLEPEDF AVYYCFQGSGYPFTFGQGTRLEIK huCAN46G4 K, Artificial SSVSY 754 CDR1 sequence huCAN46G4 K, Artificial ETS 755 CDR2 sequence huCAN46G4 K, Artificial FQGSGYPFT 756 CDR3 sequence huCAN46G4 K, Artificial EKVLTQSPATLSLSPGERATMTCSAS 757 FR1 sequence huCAN46G4 K, Artificial MHWYQQKPGTSPKLWIY 758 FR2 sequence huCAN46G4 K, Artificial KLAFGVPARFSGSGSGNSYSLTISSLEP 759 FR3 sequence EDFAVYYC huCAN46G4 K, Artificial FGQGTRLEIK 760 FR4 sequence huCAN46G4 H, Artificial EVQLLQSGAEVKKPGSSVKISCKASDY 761 variable sequence SFTGYYMHWVKQAPGQGLEWIGRIFP region YNGAASYNQNFKDKATLTVDKSSSTA YMELHSLRSEDTAVYYCTRWLRVYFD YWGQGTLVTVSS huCAN46G4 H, Artificial DYSFTGYY 762 CDR1 sequence huCAN46G4 H, Artificial IFPYNGAA 763 CDR2 sequence huCAN46G4 H, Artificial TRWLRVYFDY 764 CDR3 sequence huCAN46G4 H, Artificial EVQLLQSGAEVKKPGSSVKISCKAS 765 FR1 sequence huCAN46G4 H, Artificial MHWVKQAPGQGLEWIGR 766 FR2 sequence huCAN46G4 H, Artificial SYNQNFKDKATLTVDKSSSTAYMELH 767 FR3 sequence SLRSEDTAVYYC huCAN46G4 H, Artificial WGQGTLVTVSS 768 FR4 sequence rehuCAN46G4 K, Artificial EKVLTQSPATLSASPGERVTMSCSASSS 769 variable sequence VSYMHWYQQKPGQSPKLWIYETSKLA region FGVPARFSGSGSGTDYSLTISSMEPEDF ATYYCFQGSGYPFTFGQGTRLEIK rehuCAN46G4 K, Artificial SSVSY 770 CDR1 sequence rehuCAN46G4 K, Artificial ETS 771 CDR2 sequence rehuCAN46G4 K, Artificial FQGSGYPFT 772 CDR3 sequence rehuCAN46G4 K, Artificial EKVLTQSPATLSASPGERVTMSCSAS 773 FR1 sequence rehuCAN46G4 K, Artificial MHWYQQKPGQSPKLWIY 774 FR2 sequence rehuCAN46G4 K, Artificial KLAFGVPARFSGSGSGTDYSLTISSMEP 775 FR3 sequence EDFATYYC rehuCAN46G4 K, Artificial FGQGTRLEIK 776 FR4 sequence rehuCAN46G4 H, Artificial EVQLLQSGAEVVKPGSSVKISCKASGY 778 variable sequence SFTGYYMHWVKQAPGQGLEWIGRIFP region YNGAASYNQNFKDKATLTADKSTNTA YMELSSLRSEDSAVYYCTRWLRVYFD YWGQGTLVTVSS rehuCAN46G4 H, Artificial GYSFTGYY 779 CDR1 sequence rehuCAN46G4 H, Artificial IFPYNGAA 780 CDR2 sequence rehuCAN46G4 H, Artificial TRWLRVYFDY 781 CDR3 sequence rehuCAN46G4 H, Artificial EVQLLQSGAEVVKPGSSVKISCKAS 782 FR1 sequence rehuCAN46G4 H, Artificial MHWVKQAPGQGLEWIGR 783 FR2 sequence rehuCAN46G4 H, Artificial SYNQNFKDKATLTADKSTNTAYMELS 784 FR3 sequence SLRSEDSAVYYC rehuCAN46G4 H, Artificial WGQGTLVTVSS 785 FR4 sequence cdrCAN46G13a K, Artificial DIQMTQSPSSLSASVGDRVTITCSASSS 101 variable Sequence VSSSYLHWYQQKPGKAPKLLIYRTSTL region ASGVPSRFSGSGSGTDFTFTISSLQPEDI ATYYCQQWSGYPYTFGQGTKVEIK cdrCAN46G13a K, Mus SSVSSSY 102 CDR1 Musculus cdrCAN46G13a K, Mus RTS 103 CDR2 Musculus cdrCAN46G13a K, Mus QQWSGYPYT 104 CDR3 Musculus cdrCAN46G13a K, Homo DIQMTQSPSSLSASVGDRVTITCSAS 105 FR1 sapiens cdrCAN46G13a K, Homo LHWYQQKPGKAPKLLIY 106 FR2 sapiens cdrCAN46G13a K, Homo TLASGVPSRFSGSGSGTDFTFTISSLQPE 107 FR3 sapiens DIATYYC cdrCAN46G13a K, Mus FGQGTKVEIK 108 FR4 musculus cdrCAN46G13a H, Artificial QVQLQESGPGLVKPSQTLSLTCTVSGG 109 variable Sequence SISSDSAWNWIRQPPGKGLEWIGYISYS region GSTSYNPSLKSRVTMSVDTSKNQFSLK VNSVTAADTAVYYCARRSRVSFYFDY WGQGTLVTVSS cdrCAN46G13a H, Mus GGSISSDSA 110 CDR1 Musculus cdrCAN46G13a H, Mus ISYSGST 111 CDR2 Musculus cdrCAN46G13a H, Mus ARRSRVSFYFDY 112 CDR3 Musculus cdrCAN46G13a H, Homo QVQLQESGPGLVKPSQTLSLTCTVS 113 FR1 sapiens cdrCAN46G13a H, Homo WNWIRQPPGKGLEWIGY 114 FR2 sapiens cdrCAN46G13a H, Homo SYNPSLKSRVTMSVDTSKNQFSLKVNS 115 FR3 sapiens VTAADTAVYYC cdrCAN46G13a H, Mus WGQGTLVTVSS 116 FR4 musculus huCAN46G13a K, Artificial ENVLTQSPSSLSASVGDRVTMTCSASS 117 variable sequence SVSSSYLHWYQQKPGKSPKPLIHRTST region LASGVPSRFSGSGSGTSYSLTISSLQPED IATYYCQQWSGYPYTFGGGTKVEIK huCAN46G13a K, Mus SSVSSSY 118 CDR1 musculus huCAN46G13a K, Mus RTS 119 CDR2 musculus huCAN46G13a K, Mus QQWSGYPYT 120 CDR3 musculus huCAN46G13a K, Artificial ENVLTQSPSSLSASVGDRVTMTCSAS 121 FR1 sequence huCAN46G13a K, Artificial LHWYQQKPGKSPKPLIH 122 FR2 sequence huCAN46G13a K, Artificial TLASGVPSRFSGSGSGTSYSLTISSLQPE 123 FR3 sequence DIATYYC huCAN46G13a K, Mus FGGGTKVEIK 124 FR4 musculus huCAN46G13a H, Artificial QVQLQESGPGLVKPSQTLSLTCTVTGY 125 variable sequence SITSDSAWNWIRQFPGNNLEWMGYISY region SGSTSYNPSLKSRISITRDTSKNQFSLKV NSVTAADTAVYYCARRSRVSFYFDYW GQGTLVTVSS huCAN46G13a H, Artificial GYSITSDSA 126 CDR1 sequence huCAN46G13a H, Mus ISYSGST 127 CDR2 musculus huCAN46G13a H, Mus ARRSRVSFYFDY 128 CDR3 musculus huCAN46G13a H, Artificial QVQLQESGPGLVKPSQTLSLTCTVT 129 FR1 sequence huCAN46G13a H, Artificial WNWIRQFPGNNLEWMGY 130 FR2 sequence huCAN46G13a H, Artificial SYNPSLKSRISITRDTSKNQFSLKVNSV 131 FR3 sequence TAADTAVYYC huCAN46G13a H, Mus WGQGTLVTVSS 132 FR4 musculus rehuCAN46G13a K, Artificial ENVLTQSPSSMSASVGDRVTMTCSASS 133 variable sequence SVSSSYLHWYQQKPGKAPKPLIHRTST region LASGVPSRFSGSGSGTSYSLTISSVQPE DIATYYCQQWSGYPYTFGGGTKVEIK rehuCAN46G13a K, Mus SSVSSSY 134 CDR1 musculus rehuCAN46G13a K, Mus RTS 135 CDR2 musculus rehuCAN46G13a K, Mus QQWSGYPYT 136 CDR3 musculus rehuCAN46G13a K, Artificial ENVLTQSPSSMSASVGDRVTMTCSAS 137 FR1 sequence rehuCAN46G13a K, Artificial LHWYQQKPGKAPKPLIH 138 FR2 sequence rehuCAN46G13a K, Artificial TLASGVPSRFSGSGSGTSYSLTISSVQPE 139 FR3 sequence DIATYYC rehuCAN46G13a K, Mus FGGGTKVEIK 140 FR4 musculus rehuCAN46G13a H, Artificial QVQLQESGPGLVKPSQTLSLTCTVTGY 141 variable sequence SITSDSAWNWIRQPPGNGLEWMGYISY region SGSTSYNPSLKSRISITRDTSKNQFSLKL NSVTAADTATYYCARRSRVSFYFDYW GQGTLVTVSS rehuCAN46G13a H, Artificial GYSITSDSA 142 CDR1 sequence rehuCAN46G13a H, Mus ISYSGST 143 CDR2 musculus rehuCAN46G13a H, Mus ARRSRVSFYFDY 144 CDR3 musculus rehuCAN46G13a H, Artificial QVQLQESGPGLVKPSQTLSLTCTVT 145 FR1 sequence rehuCAN46G13a H, Artificial WNWIRQPPGNGLEWMGY 146 FR2 sequence rehuCAN46G13a H, Artificial SYNPSLKSRISITRDTSKNQFSLKLNSV 147 FR3 sequence TAADTATYYC rehuCAN46G13a H, Mus WGQGTLVTVSS 148 FR4 musculus cdrCAN46G19 K, Artificial DIQMTQSPSSLSASVGDRVTITCSASSS 149 variable sequence VTYMHWYQQKPGKAPKLLIYETSKLA region SGVPSRFSGSGSGTDYTFTISSLQPEDIA TYYCFQGSGYPFTFGQGTKVEIK cdrCAN46G19 K, Mus SSVTY 150 CDR1 musculus cdrCAN46G19 K, Mus ETS 151 CDR2 musculus cdrCAN46G19 K, Mus FQGSGYPFT 152 CDR3 musculus cdrCAN46G19 K, Homo DIQMTQSPSSLSASVGDRVTITCSAS 153 FR1 sapiens cdrCAN46G19 K, Homo MHWYQQKPGKAPKLLIY 154 FR2 sapiens cdrCAN46G19 K, Homo KLASGVPSRFSGSGSGTDYTFTISSLQP 155 FR3 sapiens EDIATYYC cdrCAN46G19 K, Homo FGQGTKVEIK 156 FR4 sapiens cdrCAN46G19 H, Artificial QVQLVQSGAEVKKPGESVKVSCKASG 157 variable sequence YTFTGYYIHWVRQAPGQGLEWMGRIF region PYNGAASYNQNFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARWLRVYFD YWGQGTTVTVSS cdrCAN46G19 H, Artificial GYTFTGYY 158 CDR1 sequence cdrCAN46G19 H, Mus IFPYNGAA 159 CDR2 musculus cdrCAN46G19 H, Mus ARWLRVYFDY 160 CDR3 musculus cdrCAN46G19 H, Homo QVQLVQSGAEVKKPGESVKVSCKAS 161 FR1 sapiens cdrCAN46G19 H, Homo IHWVRQAPGQGLEWMGR 162 FR2 sapiens cdrCAN46G19 H, Homo SYNQNFKGRVTITADKSTSTAYMELSS 163 FR3 sapiens LRSEDTAVYYC cdrCAN46G19 H, Homo WGQGTTVTVSS 164 FR4 sapiens huCAN46G19 K, Artificial ENVLTQSPSSLSASVGDRVTITCSASSS 165 variable sequence VTYMHWYQQKPGKAPKLWIYETSKL region ASGVPGRFSGSGSGNSYTFTISSLQPEDI ATYYCFQGSGYPFTFGQGTKVEIK huCAN46G19 K, Mus SSVTY 166 CDR1 musculus huCAN46G19 K, Mus ETS 167 CDR2 musculus huCAN46G19 K, Mus FQGSGYPFT 168 CDR3 musculus huCAN46G19 K, Artificial ENVLTQSPSSLSASVGDRVTITCSAS 169 FR1 sequence huCAN46G19 K, Artificial MHWYQQKPGKAPKLWIY 170 FR2 sequence huCAN46G19 K, Artificial KLASGVPGRFSGSGSGNSYTFTISSLQP 171 FR3 sequence EDIATYYC huCAN46G19 K, Homo FGQGTKVEIK 172 FR4 sapiens huCAN46G19 H, Artificial EVQLVQSGAEVKKPGESVKVSCKASG 173 variable sequence YSFTGYYIHWVKQAPGQGLEWVGRIF region PYNGAASYNQNFKGKATLTVDKSSTT AYMELSSLRSEDTAVYFCARWLRVYF DYWGQGTTVTVSS huCAN46G19 H, Mus GYSFTGYY 174 CDR1 musculus huCAN46G19 H, Mus IFPYNGAA 175 CDR2 musculus huCAN46G19 H, Mus ARWLRVYFDY 176 CDR3 musculus huCAN46G19 H, Artificial EVQLVQSGAEVKKPGESVKVSCKAS 177 FR1 sequence huCAN46G19 H, Artificial IHWVKQAPGQGLEWVGR 178 FR2 sequence huCAN46G19 H, Artificial SYNQNFKGKATLTVDKSSTTAYMELS 179 FR3 sequence SLRSEDTAVYFC huCAN46G19 H, Homo WGQGTTVTVSS 180 FR4 sapiens rehuCAN46G19 K, Artificial ENVLTQSPSSMSASVGDRVTMTCSASS 181 variable sequence SVTYMHWYQQKPGKSPKLWIYETSKL region ASGVPSRFSGSGSGNDYSLTISSMQPED VATYYCFQGSGYPFTFGQGTKLEIK rehuCAN46G19 K, Mus SSVTY 182 CDR1 musculus rehuCAN46G19 K, Mus ETS 183 CDR2 musculus rehuCAN46G19 K, Mus FQGSGYPFT 184 CDR3 musculus rehuCAN46G19 K, Artificial ENVLTQSPSSMSASVGDRVTMTCSAS 185 FR1 sequence rehuCAN46G19 K, Artificial MHWYQQKPGKSPKLWIY 186 FR2 sequence rehuCAN46G19 K, Artificial KLASGVPSRFSGSGSGNDYSLTISSMQP 187 FR3 sequence EDVATYYC rehuCAN46G19 K, Homo FGQGTKLEIK 188 FR4 sapiens rehuCAN46G19 H, Artificial EVQLVQSGAEVVKPGESVKISCKASGY 189 variable sequence SFTGYYIHWVKQTPGQSLEWVGRIFPY region NGAASYNQNFKGKATLTVDKSTTTAY MELSSLRSEDSAVYFCARWLRVYFDY WGQGTTLTVSS rehuCAN46G19 H, Mus GYSFTGYY 190 CDR1 musculus rehuCAN46G19 H, Mus IFPYNGAA 191 CDR2 musculus rehuCAN46G19 H, Mus ARWLRVYFDY 192 CDR3 musculus rehuCAN46G19 H, Artificial EVQLVQSGAEVVKPGESVKISCKAS 193 FR1 sequence rehuCAN46G19 H, Artificial IHWVKQTPGQSLEWVGR 194 FR2 sequence rehuCAN46G19 H, Artificial SYNQNFKGKATLTVDKSTTTAYMELS 195 FR3 sequence SLRSEDSAVYFC rehuCAN46G19 H, Mus WGQGTTLTVSS 196 FR4 musculus cdrCAN46G24 K, Artificial DIQMTQSPSSLSASVGDRVTITCSASSS 197 variable sequence VTYMHWYQQKPGKAPKLLIYETSKLA region SGVPSRFSGSGSGTDYTFTISSLQPEDIA TYYCFQGSGYPFTFGQGTKVEIK cdrCAN46G24 K, Mus SSVTY 198 CDR1 musculus cdrCAN46G24 K, Mus ETS 199 CDR2 musculus cdrCAN46G24 K, Mus FQGSGYPFT 200 CDR3 musculus cdrCAN46G24 K, Homo DIQMTQSPSSLSASVGDRVTITCSAS 201 FR1 sapiens cdrCAN46G24 K, Homo MHWYQQKPGKAPKLLIY 202 FR2 sapiens cdrCAN46G24 K, Homo KLASGVPSRFSGSGSGTDYTFTISSLQP 203 FR3 sapiens EDIATYYC cdrCAN46G24 K, Homo FGQGTKVEIK 204 FR4 sapiens cdrCAN46G24 H, Artificial QVQLVQSGAEVKKPGESVKVSCKASG 205 variable sequence YTFTGYYIHWVRQAPGQGLEWMGRIF region PYNGAASYNQNFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARWLRVYFD YWGQGTTVTVSS cdrCAN46G24 H, Mus GYTFTGYY 206 CDR1 musculus cdrCAN46G24 H, Mus IFPYNGAA 207 CDR2 musculus cdrCAN46G24 H, Mus ARWLRVYFDY 208 CDR3 musculus cdrCAN46G24 H, Homo QVQLVQSGAEVKKPGESVKVSCKAS 209 FR1 sapiens cdrCAN46G24 H, Homo IHWVRQAPGQGLEWMGR 210 FR2 sapiens cdrCAN46G24 H, Homo SYNQNFKGRVTITADKSTSTAYMELSS 211 FR3 sapiens LRSEDTAVYYC cdrCAN46G24 H, Homo WGQGTTVTVSS 212 FR4 sapiens huCAN46G24 K, Artificial EIVLTQSPSSLSTSVGDRVTISCSASSSV 213 variable sequence TYMHWYQQKPGKAPKLWIYETSKLAS region GVPGRFSGSGSGNSYTFTISSLQPEDIA TYYCFQGSGYPFTFGQGTKVEIK huCAN46G24 K, Mus SSVTY 214 CDR1 musculus huCAN46G24 K, Mus ETS 215 CDR2 musculus huCAN46G24 K, Mus FQGSGYPFT 216 CDR3 musculus huCAN46G24 K, Artificial EIVLTQSPSSLSTSVGDRVTISCSAS 217 FR1 sequence huCAN46G24 K, Artificial MHWYQQKPGKAPKLWIY 218 FR2 sequence huCAN46G24 K, Artificial KLASGVPGRFSGSGSGNSYTFTISSLQP 219 FR3 sequence EDIATYYC huCAN46G24 K, Homo FGQGTKVEIK 220 FR4 sapiens huCAN46G24 H, Artificial EVQLVQSGAEVKKPGESVKVSCKASG 221 variable sequence YSFTGYYIHWVKQAPGQGLEWVGRIF region PYNGAASYNQNFKGKATLTVDKSSST AYMELSSLRSEDTAVYFCARWLRVYF DYWGQGTTVTVSS huCAN46G24 H, Mus GYSFTGYY 222 CDR1 musculus huCAN46G24 H, Mus IFPYNGAA 223 CDR2 musculus huCAN46G24 H, Mus ARWLRVYFDY 224 CDR3 musculus huCAN46G24 H, Artificial EVQLVQSGAEVKKPGESVKVSCKAS 225 FR1 sequence huCAN46G24 H, Artificial IHWVKQAPGQGLEWVGR 226 FR2 sequence huCAN46G24 H, Artificial SYNQNFKGKATLTVDKSSSTAYMELSS 227 FR3 sequence LRSEDTAVYFC huCAN46G24 H, Homo WGQGTTVTVSS 228 FR4 sapiens rehuCAN46G24 K, Artificial EIVLTQSPSSMSTSVGDRVTMSCSASSS 229 variable sequence VTYMHWYQQKPGKSPKLWIYETSKLA region SGVPSRFSGSGSGNDYSLTISSMQPEDV ATYYCFQGSGYPFTFGQGTKLEIK rehuCAN46G24 K, Mus SSVTY 230 CDR1 musculus rehuCAN46G24 K, Mus ETS 231 CDR2 musculus rehuCAN46G24 K, Mus FQGSGYPFT 232 CDR3 musculus rehuCAN46G24 K, Artificial EIVLTQSPSSMSTSVGDRVTMSCSAS 233 FR1 sequence rehuCAN46G24 K, Artificial MHWYQQKPGKSPKLWIY 234 FR2 sequence rehuCAN46G24 K, Artificial KLASGVPSRFSGSGSGNDYSLTISSMQP 235 FR3 sequence EDVATYYC rehuCAN46G24 K, Artificial FGQGTKLEIK 236 FR4 sequence rehuCAN46G24 H, Artificial EVQLVQSGAEVVKPGESVKISCKASGY 237 variable sequence SFTGYYIHWVKQTPGQSLEWVGRIFPY region NGAASYNQNFKGKATLTVDKSTSTAY MELSSLRSEDSAVYFCARWLRVYFDY WGQGTTLTVSS rehuCAN46G24 H, Artificial GYSFTGYY 238 CDR1 sequence rehuCAN46G24 H, Mus IFPYNGAA 239 CDR2 musculus rehuCAN46G24 H, Artificial ARWLRVYFDY 240 CDR3 sequence rehuCAN46G24 H, Artificial EVQLVQSGAEVVKPGESVKISCKAS 241 FR1 sequence rehuCAN46G24 H, Artificial IHWVKQTPGQSLEWVGR 242 FR2 sequence rehuCAN46G24 H, Artificial SYNQNFKGKATLTVDKSTSTAYMELS 243 FR3 sequence SLRSEDSAVYFC rehuCAN46G24 H, Mus WGQGTTLTVSS 244 FR4 musculus CAN46G4 K, Mus gaaaaggttctcacccagtctccagcaatcatgtctgcatctc 245 variable musculus caggggaagaggtcaccatgacctgcagtgccagctcaag region tgtaagttacatgcattggtaccagcagaagtcaagcacctc ccccaaactctggatttatgaaacatccaaactggcttttgga gtcccaggtcgcttcagtggcagtggatctggaaactcttact ctctcacgatcagcagcatggaggctgaagatgttgccactt attactgttttcaggggagtgggtacccattcacgttcggctc ggggacaaagttggaagtaaaa CAN46G4 K, Mus tcaagtgtaagttac 246 CDR1 musculus CAN46G4 K, Mus gaaacatcc 247 CDR2 musculus CAN46G4 K, Mus tttcaggggagtgggtacccattcacg 248 CDR3 musculus CAN46G4 K, Mus gaaaaggttctcacccagtctccagcaatcatgtctgcatctc 249 FR1 musculus caggggaagaggtcaccatgacctgcagtgccagc CAN46G4 K, Mus atgcattggtaccagcagaagtcaagcacctcccccaaact 250 FR2 musculus ctggatttat CAN46G4 K, Mus aaactggcttttggagtcccaggtcgcttcagtggcagtgga 251 FR3 musculus tctggaaactcttactctctcacgatcagcagcatggaggctg aagatgttgccacttattactgt CAN46G4 K, Mus ttcggctcggggacaaagttggaagtaaaa 252 FR4 musculus CAN46G4 H, Mus gaggtccagctgctacagtctggccctgagctggtgaagcc 253 variable musculus tggggcttcagtgaagatatcctgcaaggcttctgattactcat region tcactggctactacatgcactgggtgaagcaaagccatgtaa agagccttgagtggattggacgtatttttccttacaatggtgct gctagctacaaccagaatttcaaggacaaggccaccttgact gtagataagtcttccagcacagcctacatggagctccacagc ctgacatctgaggactctgcagtctattattgtacaagatggtt aagggtctactttgactactggggccaaggcaccactctcac agtctcctca CAN46G4 H, Mus gattactcattcactggctactac 254 CDR1 musculus CAN46G4 H, Mus atttttccttacaatggtgctgct 255 CDR2 musculus CAN46G4 H, Mus acaagatggttaagggtctactttgactac 256 CDR3 musculus CAN46G4 H, Mus gaggtccagctgctacagtctggccctgagctggtgaagcc 257 FR1 musculus tggggcttcagtgaagatatcctgcaaggcttct CAN46G4 H, Mus atgcactgggtgaagcaaagccatgtaaagagccttgagtg 258 FR2 musculus gattggacgt CAN46G4 H, Mus agctacaaccagaatttcaaggacaaggccaccttgactgta 259 FR3 musculus gataagtcttccagcacagcctacatggagctccacagcctg acatctgaggactctgcagtctattattgt CAN46G4 H, Mus tggggccaaggcaccactctcacagtctcctca 260 FR4 musculus CAN46G13 K, Mus gaaattgttctcacccagtctccagcaatcatgtctacatctcc 261 variable musculus aggggaaaaggtcaccatgtcctgcagtgccagctcaagtg region taacttacatgcactggtaccagcagaagtcaatcacctccc ccaaactctggatttatgaaacatccaaactggcttctggagt ccccggtcgcttcagtggcagtgggtctggaaactcttactct ctcacgatcagcagcatggaggctgaagatgttgccacttat tactgttttcaggggagtgggtacccattcacgttcggctcgg ggacaaagttggaaataaaac CAN46G13 K, Mus tcaagtgtaacttac 262 CDR1 musculus CAN46G13 K, Mus gaaacatcc 263 CDR2 musculus CAN46G13 K, Mus tttcaggggagtgggtacccattcacg 264 CDR3 musculus CAN46G13 K, Mus gaaattgttctcacccagtctccagcaatcatgtctacatctcc 265 FR1 musculus aggggaaaaggtcaccatgtcctgcagtgccagc CAN46G13 K, Mus atgcactggtaccagcagaagtcaatcacctcccccaaactc 266 FR2 musculus tggatttat CAN46G13 K, Mus aaactggcttctggagtccccggtcgcttcagtggcagtggg 267 FR3 musculus tctggaaactcttactctctcacgatcagcagcatggaggctg aagatgttgccacttattactgt CAN46G13 K, Mus ttcggctcggggacaaagttggaaataaaac 268 FR4 musculus CAN46G13 H, Mus gaggtccagctgctacagtctggccctgagctggtgaagcc 269 variable musculus tgggacttcagtgaagatatcctgcaaggcttctggttactcat region tcactggctactacatacactgggtgaagcagacccatgtaa agagccttgagtgggttggacgtatttttccttacaatggtgct gctagctacaatcagaatttcaagggcaaggccaccttgact gtagataagtcctccagcacagcctacatggagctccacag cctgacatctgaggactctgcagtctatttctgtgcaagatggt taagggtctactttgactactggggccaaggcaccactctca cagtctcctcag CAN46G13 H, Mus ggttactcattcactggctactac 270 CDR1 musculus CAN46G13 H, Mus atttttccttacaatggtgctgct 271 CDR2 musculus CAN46G13 H, Mus gcaagatggttaagggtctactttgactac 272 CDR3 musculus CAN46G13 H, Mus gaggtccagctgctacagtctggccctgagctggtgaagcc 273 FR1 musculus tgggacttcagtgaagatatcctgcaaggcttct CAN46G13 H, Mus atacactgggtgaagcagacccatgtaaagagccttgagtg 274 FR2 musculus ggttggacgt CAN46G13 H, Mus agctacaatcagaatttcaagggcaaggccaccttgactgta 275 FR3 musculus gataagtcctccagcacagcctacatggagctccacagcct gacatctgaggactctgcagtctatttctgt CAN46G13 H, Mus tggggccaaggcaccactctcacagtctcctcag 276 FR4 musculus CAN46G13a K, Mus gaaaatgtgctcacccagtctccagcaataatggctgcctct 277 variable musculus ctggggcagaaggtcaccatgacctgcagtgccagctcaa region gtgtaagttccagttacttgcactggtaccagcagaagtcag gcgcttcccccaaacccttgattcataggacatccaccctgg cttctggcgtcccagctcgcttcagtggcagtgggtctggga cctcttactctctcacaatcagcagcgtggaggctgaagatg atgcaacttattactgccagcagtggagtggttacccgtacac gttcggaggggggaccaagctggaaataaaa CAN46G13a K, Mus tcaagtgtaagttccagttac 278 CDR1 musculus CAN46G13a K, Mus aggacatcc 279 CDR2 musculus CAN46G13a K, Mus cagcagtggagtggttacccgtacacg 280 CDR3 musculus CAN46G13a K, Mus gaaaatgtgctcacccagtctccagcaataatggctgcctct 281 FR1 musculus ctggggcagaaggtcaccatgacctgcagtgccagc CAN46G13a K, Mus ttgcactggtaccagcagaagtcaggcgcttcccccaaacc 282 FR2 musculus cttgattcat CAN46G13a K, Mus accctggcttctggcgtcccagctcgcttcagtggcagtggg 283 FR3 musculus tctgggacctcttactctctcacaatcagcagcgtggaggct gaagatgatgcaacttattactgc CAN46G13a K, Mus ttcggaggggggaccaagctggaaataaaa 284 FR4 musculus CAN46G13a H, Mus gatgtgcagcttcaggagtcaggacctggcctggtgaaacc 285 variable musculus ttctcagtctctgtccctcacctgcactgtcactggctactcaat region caccagtgattctgcctggaactggatccggcagtttccagg aaacaacctggagtggatgggctacataagctacagtggta gcactagctacaacccatctctcaaaagtcgaatctctatcac tcgagacacatccaagaaccagttcttcctgcagttgaattct gtgactactgaggacacagccacatattactgtgcaagaag gagtagggtctcattctactttgactactggggccaaggcac cactctcacagtctcctcag CAN46G13a H, Mus ggctactcaatcaccagtgattctgcc 286 CDR1 musculus CAN46G13a H, Mus ataagctacagtggtagcact 287 CDR2 musculus CAN46G13a H, Mus gcaagaaggagtagggtctcattctactttgactac 288 CDR3 musculus CAN46G13a H, Mus gatgtgcagcttcaggagtcaggacctggcctggtgaaacc 289 FR1 musculus ttctcagtctctgtccctcacctgcactgtcact CAN46G13a H, Mus tggaactggatccggcagtttccaggaaacaacctggagtg 290 FR2 musculus gatgggctac CAN46G13a H, Mus agctacaacccatctctcaaaagtcgaatctctatcactcgag 291 FR3 musculus acacatccaagaaccagttcttcctgcagttgaattctgtgact actgaggacacagccacatattactgt CAN46G13a H, Mus tggggccaaggcaccactctcacagtctcctcag 292 FR4 musculus CAN46G19 K, Mus gaaaatgttctcacccagtctccaacaatcatgtctgcatctcc 293 variable musculus aggggaagaggtcaccatgacctgcagtgccagctcaagt region gtaacttacatgcactggtaccagcagaagtcaatcacctcc cccaaactctggatttatgaaacatccaaactggcttctggag tcccaggtcgcttcagtggcagtgggtctggaaactcttactc tctcacgatcagcagcatggaggctgaagatgttgccactta ttactgttttcaggggagtgggtacccattcacgttcggctcg gggacaaagttggaaataaaac CAN46G19 K, Mus tcaagtgtaacttac 294 CDR1 musculus CAN46G19 K, Mus gaaacatcc 295 CDR2 musculus CAN46G19 K, Mus tttcaggggagtgggtacccattcacg 296 CDR3 musculus CAN46G19 K, Mus gaaaatgttctcacccagtctccaacaatcatgtctgcatctcc 297 FR1 musculus aggggaagaggtcaccatgacctgcagtgccagc CAN46G19 K, Mus atgcactggtaccagcagaagtcaatcacctcccccaaactc 298 FR2 musculus tggatttat CAN46G19 K, Mus aaactggcttctggagtcccaggtcgcttcagtggcagtggg 299 FR3 musculus tctggaaactcttactctctcacgatcagcagcatggaggctg aagatgttgccacttattactgt CAN46G19 K, Mus ttcggctcggggacaaagttggaaataaaac 300 FR4 musculus CAN46G19 H, Mus gaggtccagctgctacagtctggccctgagctggtgaagcc 301 variable musculus tgggacttcagtgaagatatcctgcaaggcttctggttactcat region tcactggctactacattcactgggtgaagcagacccatgtaa agagccttgagtgggttggacgtatttttccttacaatggtgct gctagctacaaccagaatttcaagggcaaggccaccttgact gtagataagtcctccaccacagcctacatggagctccacag cctgacatctgaggactctgcagtctatttctgtgcaagatggt taagggtctactttgactactggggccaaggcaccactctca cagtctcctcag CAN46G19 H, Mus ggttactcattcactggctactac 302 CDR1 musculus CAN46G19 H, Mus atttttccttacaatggtgctgct 303 CDR2 musculus CAN46G19 H, Mus gcaagatggttaagggtctactttgactac 304 CDR3 musculus CAN46G19 H, Mus gaggtccagctgctacagtctggccctgagctggtgaagcc 305 FR1 musculus tgggacttcagtgaagatatcctgcaaggcttct CAN46G19 H, Mus attcactgggtgaagcagacccatgtaaagagccttgagtg 306 FR2 musculus ggttggacgt CAN46G19 H, Mus agctacaaccagaatttcaagggcaaggccaccttgactgta 307 FR3 musculus gataagtcctccaccacagcctacatggagctccacagcct gacatctgaggactctgcagtctatttctgt CAN46G19 H, Mus tggggccaaggcaccactctcacagtctcctcag 308 FR4 musculus CAN46G24 K, Mus gaaattgttctcacccagtctccagcaatcatgtctacatctcc 309 variable musculus aggggaaaaggtcaccatgtcctgcagtgccagctcaagtg region taacttacatgcactggtaccagcagaagtcaatcacctccc ccaaactctggatttatgaaacatccaaactggcttctggagt ccccggtcgcttcagtggcagtgggtctggaaactcttactct ctcacgatcagcagcatggaggctgaagatgttgccacttat tactgttttcaggggagtgggtacccattcacgttcggctcgg ggacaaagttggaaataaaac CAN46G24 K, Mus tcaagtgtaacttac 310 CDR1 musculus CAN46G24 K, Mus gaaacatcc 311 CDR2 musculus CAN46G24 K, Mus tttcaggggagtgggtacccattcacg 312 CDR3 musculus CAN46G24 K, Mus gaaattgttctcacccagtctccagcaatcatgtctacatctcc 313 FR1 musculus aggggaaaaggtcaccatgtcctgcagtgccagc CAN46G24 K, Mus atgcactggtaccagcagaagtcaatcacctcccccaaactc 314 FR2 musculus tggatttat CAN46G24 K, Mus aaactggcttctggagtccccggtcgcttcagtggcagtggg 315 FR3 musculus tctggaaactcttactctctcacgatcagcagcatggaggctg aagatgttgccacttattactgt CAN46G24 K, Mus ttcggctcggggacaaagttggaaataaaac 316 FR4 musculus CAN46G24 H, Mus gaggtccagctgctacagtctggccctgagctggtgaagcc 317 variable musculus tgggacttcagtgaagatatcctgcaaggcttctggttactcat region tcactggctactacatacactgggtgaagcagacccatgtaa agagccttgagtgggttggacgtatttttccttacaatggtgct gctagctacaatcagaatttcaagggcaaggccaccttgact gtagataagtcctccagcacagcctacatggagctccacag cctgacatctgaggactctgcagtctatttctgtgcaagatggt taagggtctactttgactactggggccaaggcaccactctca cagtctcctcag CAN46G24 H, Mus ggttactcattcactggctactac 318 CDR1 musculus CAN46G24 H, Mus atttttccttacaatggtgctgct 319 CDR2 musculus CAN46G24 H, Mus gcaagatggttaagggtctactttgactac 320 CDR3 musculus CAN46G24 H, Mus gaggtccagctgctacagtctggccctgagctggtgaagcc 321 FR1 musculus tgggacttcagtgaagatatcctgcaaggcttct CAN46G24 H, Mus atacactgggtgaagcagacccatgtaaagagccttgagtg 322 FR2 musculus ggttggacgt CAN46G24 H, Mus agctacaatcagaatttcaagggcaaggccaccttgactgta 323 FR3 musculus gataagtcctccagcacagcctacatggagctccacagcct gacatctgaggactctgcagtctatttctgt CAN46G24 H, Mus tggggccaaggcaccactctcacagtctcctcag 324 FR4 musculus CAN33G1 K, Mus gacatccagctgacacaatcttcatcctcctattctgtatctcta 721 variable musculus ggagacagggtcaccattacttgcaaggcaagtgaggacat region atataatcggttagcctggtatcagcagagaccaggaaatgc tcctaggctcttaatatctggtgcaaccagtttggaaactggg attccttcaagattcagtggcagtggatctggaaaggagtac actctcagcattgccagtcttcagactgaagattttgttacttatt actgtcaacaatattggaatattccgacgttcggtggaggca ccaggctggaaatcaaac CAN33G1 K, Mus gaggacatatataatcgg 722 CDR1 musculus CAN33G1 K, Mus ggtgcaacc 723 CDR2 musculus CAN33G1 K, Mus caacaatattggaatattccgacg 724 CDR3 musculus CAN33G1 K, Mus gacatccagctgacacaatcttcatcctcctattctgtatctcta 725 FR1 musculus ggagacagggtcaccattacttgcaaggcaagt CAN33G1 K, Mus ttagcctggtatcagcagagaccaggaaatgctcctaggctc 726 FR2 musculus ttaatatct CAN33G1 K, Mus agtttggaaactgggattccttcaagattcagtggcagtggat 727 FR3 musculus ctggaaaggagtacactctcagcattgccagtcttcagactg aagattttgttacttattactgt CAN33G1 K, Mus ttcggtggaggcaccaggctggaaatcaaac 728 FR4 musculus CAN33G1 H, Mus gaggtccagctgcagcagtctggacctgacctggtgaagcc 729 variable musculus tggggcttcagtgaagatatcctgcaaggcttctggttactca region ttcactggctactacatgcactgggtgaagcagagccatgga aagagccttgagtggattggacgtgttaatccttacaacggtg atactaattacaaccagaatttcaaggacaaggccatattaac tgtagacaagtcagccagtacagcctacatggagttccgca gcctgacatctgaggactctgcggtctattactgtacaagatc aaactgggaaaactactttgactactggggccaaggctcca ctctcacagtctcctcag CAN33G1 H, Mus ggttactcattcactggctactac 730 CDR1 musculus CAN33G1 H, Mus gttaatccttacaacggtgatact 731 CDR2 musculus CAN33G1 H, Mus acaagatcaaactgggaaaactactttgactac 732 CDR3 musculus CAN33G1 H, Mus gaggtccagctgcagcagtctggacctgacctggtgaagcc 733 FR1 musculus tggggcttcagtgaagatatcctgcaaggcttct CAN33G1 H, Mus atgcactgggtgaagcagagccatggaaagagccttgagt 734 FR2 musculus ggattggacgt CAN33G1 H, Mus aattacaaccagaatttcaaggacaaggccatattaactgtag 735 FR3 musculus acaagtcagccagtacagcctacatggagttccgcagcctg acatctgaggactctgcggtctattactgt CAN33G1 H, Mus tggggccaaggctccactctcacagtctcctcag 736 FR4 musculus CAN46G4 K, Artificial gaaaaggttctcacccagtctccagcaatcatgtctgcatctc 325 Codon  variable sequence caggggaagaggtcaccatgacctgcagtgccagctcaag optimized region tgtaagttacatgcattggtaccagcagaagtcaagcacctc ccccaaactctggatttatgaaacatccaaactggcttttgga gtcccaggtcgcttcagtggcagtggatctggaaactcttact ctctcacgatcagcagcatggaggctgaagatgttgccactt attactgttttcaggggagtgggtacccattcacgttcggctc ggggacaaagttggaagtaaaac CAN46G4 Codon K, Artificial tcaagtgtaagttac 326 optimized CDR1 sequence CAN46G4 Codon K, Artificial gaaacatcc 327 optimized CDR2 sequence CAN46G4 Codon K, Artificial tttcaggggagtgggtacccattcacg 328 optimized CDR3 sequence CAN46G4 Codon K, Artificial gaaaaggttctcacccagtctccagcaatcatgtctgcatctc 329 optimized FR1 sequence caggggaagaggtcaccatgacctgcagtgccagc CAN46G4 Codon K, Artificial atgcattggtaccagcagaagtcaagcacctcccccaaact 330 optimized FR2 sequence ctggatttat CAN46G4 Codon K, Artificial aaactggcttttggagtcccaggtcgcttcagtggcagtgga 331 optimized FR3 sequence tctggaaactcttactctctcacgatcagcagcatggaggctg aagatgttgccacttattactgt CAN46G4 Codon K, Artificial ttcggctcggggacaaagttggaagtaaaac 332 optimized FR4 sequence CAN46G13a K, Artificial gaaaatgtgctcacccagtctccagcaataatggctgcctct 333 Codon  variable sequence ctggggcagaaggtcaccatgacctgcagtgccagctcaa optimized region gtgtaagttccagttacttgcactggtaccagcagaagtcag gcgcttcccccaaacccttgattcataggacatccaccctgg cttctggcgtcccagctcgcttcagtggcagtgggtctggga cctcttactctctcacaatcagcagcgtggaggctgaagatg atgcaacttattactgccagcagtggagtggttacccgtacac gttcggaggggggaccaagctggaaataaaac CAN46G13a K, Artificial tcaagtgtaagttccagttac 334 Codon  CDR1 sequence optimized CAN46G13a K, Artificial aggacatcc 335 Codon  CDR2 sequence optimized CAN46G13a K, Artificial cagcagtggagtggttacccgtacacg 336 Codon  CDR3 sequence optimized CAN46G13a K, Artificial gaaaatgtgctcacccagtctccagcaataatggctgcctct 337 Codon  FR1 sequence ctggggcagaaggtcaccatgacctgcagtgccagc optimized CAN46G13a K, Artificial ttgcactggtaccagcagaagtcaggcgcttcccccaaacc 338 Codon  FR2 sequence cttgattcat optimized CAN46G13a K, Artificial accctggcttctggcgtcccagctcgcttcagtggcagtggg 339 Codon  FR3 sequence tctgggacctcttactctctcacaatcagcagcgtggaggct optimized gaagatgatgcaacttattactgc CAN46G13a K, Artificial ttcggaggggggaccaagctggaaataaaac 340 Codon  FR4 sequence optimized CAN46G13a H, Artificial gatgtgcagcttcaggagtcaggacctggcctggtgaaacc 341 Codon  variable sequence ttctcagtctctgtccctcacctgcactgtcactggctactcaat optimized region caccagtgattctgcctggaactggattcggcagtttccagg aaacaacctggagtggatgggctacataagctacagtggta gcactagctacaacccatctctcaaaagtcgaatctctatcac tcgagacacatccaagaaccagttcttcctgcagttgaactct gtgactactgaggacacagccacatattactgtgcaagaag gagtagggtctcattctactttgactactggggccaaggcac cactctcacagtctcctcag CAN46G13a H, Artificial ggctactcaatcaccagtgattctgcc 342 Codon  CDR1 sequence optimized CAN46G13a H, Artificial ataagctacagtggtagcact 343 Codon  CDR2 sequence optimized CAN46G13a H, Artificial gcaagaaggagtagggtctcattctactttgactac 344 Codon  CDR3 sequence optimized CAN46G13a H, Artificial gatgtgcagcttcaggagtcaggacctggcctggtgaaacc 345 Codon  FR1 sequence ttctcagtctctgtccctcacctgcactgtcact optimized CAN46G13a H, Artificial tggaactggattcggcagtttccaggaaacaacctggagtg 346 Codon  FR2 sequence gatgggctac optimized CAN46G13a H, Artificial agctacaacccatctctcaaaagtcgaatctctatcactcgag 347 Codon  FR3 sequence acacatccaagaaccagttcttcctgcagttgaactctgtgac optimized tactgaggacacagccacatattactgt CAN46G13a H, Artificial tggggccaaggcaccactctcacagtctcctcag 348 Codon  FR4 sequence optimized CAN46G19 K, Artificial gaaaatgttctcacccagtctccaacaatcatgtctgcatctcc 349 Codon  variable sequence aggggaagaggtcaccatgacctgcagtgccagctcaagt optimized region gtaacttacatgcactggtaccagcagaagtcaatcacctcc cccaaactctggatttatgaaacatccaaactggcttctggag tcccaggtcgcttcagtggcagtgggtctggaaactcttactc tctcacgatcagcagcatggaggctgaagatgttgccactta ttactgttttcaggggagtgggtacccattcacgttcggctcg gggacaaagttggaaataaaac CAN46G19 K, Artificial tcaagtgtaacttac 350 Codon  CDR1 sequence optimized CAN46G19 K, Artificial gaaacatcc 351 Codon  CDR2 sequence optimized CAN46G19 K, Artificial tttcaggggagtgggtacccattcacg 352 Codon  CDR3 sequence optimized CAN46G19 K, Artificial gaaaatgttctcacccagtctccaacaatcatgtctgcatctcc 353 Codon  FR1 sequence aggggaagaggtcaccatgacctgcagtgccagc optimized CAN46G19 K, Artificial atgcactggtaccagcagaagtcaatcacctcccccaaactc 354 Codon  FR2 sequence tggatttat optimized CAN46G19 K, Artificial aaactggcttctggagtcccaggtcgcttcagtggcagtggg 355 Codon  FR3 sequence tctggaaactcttactctctcacgatcagcagcatggaggctg optimized aagatgttgccacttattactgt CAN46G19 K, Artificial ttcggctcggggacaaagttggaaataaaac 356 Codon  FR4 sequence optimized CAN46G19 H, Artificial gaggtccagctgctacagtctggccctgagctggtgaagcc 357 Codon  variable sequence tgggacttcagtgaagatatcctgcaaggcttctggttactcat optimized region tcactggctactacattcactgggtgaagcagacccatgtaa agagccttgagtgggttggacgtatttttccttacaatggtgct gcaagctacaaccagaatttcaagggcaaggccaccttgac tgtagataagtcctccaccacagcctacatggagctccacag cctgacatctgaggactctgcagtctatttctgtgcaagatggt taagggtctactttgactactggggccaaggcaccactctca cagtctcctcag CAN46G19 H, Artificial ggttactcattcactggctactac 358 Codon  CDR1 sequence optimized CAN46G19 H, Artificial atttttccttacaatggtgctgca 359 Codon  CDR2 sequence optimized CAN46G19 H, Artificial gcaagatggttaagggtctactttgactac 360 Codon  CDR3 sequence optimized CAN46G19 H, Artificial gaggtccagctgctacagtctggccctgagctggtgaagcc 361 Codon  FR1 sequence tgggacttcagtgaagatatcctgcaaggcttct optimized CAN46G19 H, Artificial attcactgggtgaagcagacccatgtaaagagccttgagtg 362 Codon  FR2 sequence ggttggacgt optimized CAN46G19 H, Artificial agctacaaccagaatttcaagggcaaggccaccttgactgta 363 Codon  FR3 sequence gataagtcctccaccacagcctacatggagctccacagcct optimized gacatctgaggactctgcagtctatttctgt CAN46G19 H, Artificial tggggccaaggcaccactctcacagtctcctcag 364 Codon  FR4 sequence optimized CAN46G24 K, Artificial gaaattgttctcacccagtctccagcaatcatgtctacatctcc 365 Codon  variable sequence aggggaaaaggtcaccatgtcctgcagtgccagctcaagtg optimized region taacttacatgcactggtaccagcagaagtcaatcacctccc ccaaactctggatttatgaaacatccaaactggcttctggagt ccccggtcgcttcagtggcagtgggtctggaaactcttactct ctcacgatcagcagcatggaggctgaagatgttgccacttat tactgttttcaggggagtgggtacccattcacgttcggctcgg ggacaaagttggaaataaaac CAN46G24 K, Artificial tcaagtgtaacttac 366 Codon  CDR1 sequence optimized CAN46G24 K, Artificial gaaacatcc 367 Codon  CDR2 sequence optimized CAN46G24 K, Artificial tttcaggggagtgggtacccattcacg 368 Codon  CDR3 sequence optimized CAN46G24 K, Artificial gaaattgttctcacccagtctccagcaatcatgtctacatctcc 369 Codon  FR1 sequence aggggaaaaggtcaccatgtcctgcagtgccagc optimized CAN46G24 K, Artificial atgcactggtaccagcagaagtcaatcacctcccccaaactc 370 Codon  FR2 sequence tggatttat optimized CAN46G24 K, Artificial aaactggcttctggagtccccggtcgcttcagtggcagtggg 371 Codon  FR3 sequence tctggaaactcttactctctcacgatcagcagcatggaggctg optimized aagatgttgccacttattactgt CAN46G24 K, Artificial ttcggctcggggacaaagttggaaataaaac 372 Codon  FR4 sequence optimized CAN46G24 H, Artificial gaggtccagctgctacagtctggccctgagctggtgaagcc 373 Codon  variable sequence tgggacttcagtgaagatatcctgcaaggcttctggttactcat optimized region tcactggctactacatacactgggtgaagcagacccatgtaa agagccttgagtgggttggacgtatttttccttacaatggtgct gctagctacaatcagaatttcaagggcaaggccaccttgact gtagataagtcctccagcacagcctacatggagctccacag cctgacatctgaggactctgcagtctatttctgtgcaagatggt taagggtctactttgactactggggccaaggcaccactctca cagtctcctcag CAN46G24 H, Artificial ggttactcattcactggctactac 374 Codon  CDR1 sequence optimized CAN46G24 H, Artificial atttttccttacaatggtgctgct 375 Codon  CDR2 sequence optimized CAN46G24 H, Artificial gcaagatggttaagggtctactttgactac 376 Codon  CDR3 sequence optimized CAN46G24 H, Artificial gaggtccagctgctacagtctggccctgagctggtgaagcc 377 Codon  FR1 sequence tgggacttcagtgaagatatcctgcaaggcttct optimized CAN46G24 H, Artificial atacactgggtgaagcagacccatgtaaagagccttgagtg 378 Codon  FR2 sequence ggttggacgt optimized CAN46G24 H, Artificial agctacaatcagaatttcaagggcaaggccaccttgactgta 379 Codon  FR3 sequence gataagtcctccagcacagcctacatggagctccacagcct optimized gacatctgaggactctgcagtctatttctgt CAN46G24 H, Artificial tggggccaaggcaccactctcacagtctcctcag 380 Codon  FR4 sequence optimized cdrCAN46G13a K, Artificial gacatccagatgacccagtccccctcctccctgtccgcctcc 381 variable sequence gtgggcgaccgcgtgaccatcacctgctccgcctcctcctc region cgtgtcctcctcctacctgcactggtaccagcagaagcccg gcaaggcccccaagctgctgatctaccgcacctccaccctg gcctccggcgtgccctcccgcttctccggctccggctccgg caccgacttcaccttcaccatctcctccctgcagcccgagga catcgccacctactactgccagcagtggtccggctaccccta caccttcggccagggcaccaaggtggagatcaagc cdrCAN46G13a K, Artificial tcctccgtgtcctcctcctac 382 CDR1 sequence cdrCAN46G13a K, Artificial cgcacctcc 383 CDR2 sequence cdrCAN46G13a K, Artificial cagcagtggtccggctacccctacacc 384 CDR3 sequence cdrCAN46G13a K, Artificial gacatccagatgacccagtccccctcctccctgtccgcctcc 385 FR1 sequence gtgggcgaccgcgtgaccatcacctgctccgcctcc cdrCAN46G13a K, Artificial ctgcactggtaccagcagaagcccggcaaggcccccaag 386 FR2 sequence ctgctgatctac cdrCAN46G13a K, Artificial accctggcctccggcgtgccctcccgcttctccggctccgg 387 FR3 sequence ctccggcaccgacttcaccttcaccatctcctccctgcagcc cgaggacatcgccacctactactgc cdrCAN46G13a K, Artificial ttcggccagggcaccaaggtggagatcaagc 388 FR4 sequence cdrCAN46G13a H, Artificial caggtgcagctgcaggagtccggccccggcctggtgaagc 389 variable sequence cctcccagaccctgtccctgacctgcaccgtgtccggcggc region tccatctcctccgactccgcctggaactggatccgccagccc cccggcaagggcctggagtggatcggctacatctcctactc cggctccacctcctacaacccctccctgaagtcccgcgtga ccatgtccgtggacacctccaagaaccagttctccctgaagg tgaactccgtgaccgccgccgacaccgccgtgtactactgc gcccgccgctcccgcgtgtccttctacttcgactactggggc cagggcaccctggtgaccgtgtcctccg cdrCAN46G13a H, Artificial ggcggctccatctcctccgactccgcc 390 CDR1 sequence cdrCAN46G13a H, Artificial atctcctactccggctccacc 391 CDR2 sequence cdrCAN46G13a H, Artificial gcccgccgctcccgcgtgtccttctacttcgactac 392 CDR3 sequence cdrCAN46G13a H, Artificial caggtgcagctgcaggagtccggccccggcctggtgaagc 393 FR1 sequence cctcccagaccctgtccctgacctgcaccgtgtcc cdrCAN46G13a H, Artificial tggaactggatccgccagccccccggcaagggcctggagt 394 FR2 sequence ggatcggctac cdrCAN46G13a H, Artificial tcctacaacccctccctgaagtcccgcgtgaccatgtccgtg 395 FR3 sequence gacacctccaagaaccagttctccctgaaggtgaactccgtg accgccgccgacaccgccgtgtactactgc cdrCAN46G13a H, Artificial tggggccagggcaccctggtgaccgtgtcctccg 396 FR4 sequence huCAN46G13a K, Artificial gagaacgtgctgacccagtccccctcctccctgtccgcctcc 397 variable sequence gtgggcgaccgcgtgaccatgacctgctccgcctcctcctc region cgtgtcctcctcctacctgcactggtaccagcagaagcccg gcaagtcccccaagcccctgatccaccgcacctccaccctg gcctccggcgtgccctcccgcttctccggctccggctccgg cacctcctactccctgaccatctcctccctgcagcccgagga catcgccacctactactgccagcagtggtccggctaccccta caccttcggcggcggcaccaaggtggagatcaagc huCAN46G13a K, Artificial tcctccgtgtcctcctcctac 398 CDR1 sequence huCAN46G13a K, Artificial cgcacctcc 399 CDR2 sequence huCAN46G13a K, Artificial cagcagtggtccggctacccctacacc 400 CDR3 sequence huCAN46G13a K, Artificial gagaacgtgctgacccagtccccctcctccctgtccgcctcc 401 FR1 sequence gtgggcgaccgcgtgaccatgacctgctccgcctcc huCAN46G13a K, Artificial ctgcactggtaccagcagaagcccggcaagtcccccaagc 402 FR2 sequence ccctgatccac huCAN46G13a K, Artificial accctggcctccggcgtgccctcccgcttctccggctccgg 403 FR3 sequence ctccggcacctcctactccctgaccatctcctccctgcagcc cgaggacatcgccacctactactgc huCAN46G13a K, Artificial ttcggcggcggcaccaaggtggagatcaagc 404 FR4 sequence huCAN46G13a H, Artificial caggtgcagctgcaggagtccggccccggcctggtgaagc 405 variable sequence cctcccagaccctgtccctgacctgcaccgtgaccggctact region ccatcacctccgactccgcctggaactggatccgccagttcc ccggcaacaacctggagtggatgggctacatctcctactcc ggctccacctcctacaacccctccctgaagtcccgcatctcc atcacccgcgacacctccaagaaccagttctccctgaaggt gaactccgtgaccgccgccgacaccgccgtgtactactgcg cccgccgctcccgcgtgtccttctacttcgactactggggcc agggcaccctggtgaccgtgtcctccg huCAN46G13a H, Artificial ggctactccatcacctccgactccgcc 406 CDR1 sequence huCAN46G13a H, Artificial atctcctactccggctccacc 407 CDR2 sequence huCAN46G13a H, Artificial gcccgccgctcccgcgtgtccttctacttcgactac 408 CDR3 sequence huCAN46G13a H, Artificial caggtgcagctgcaggagtccggccccggcctggtgaagc 409 FR1 sequence cctcccagaccctgtccctgacctgcaccgtgacc huCAN46G13a H, Artificial tggaactggatccgccagttccccggcaacaacctggagtg 410 FR2 sequence gatgggctac huCAN46G13a H, Artificial tcctacaacccctccctgaagtcccgcatctccatcacccgc 411 FR3 sequence gacacctccaagaaccagttctccctgaaggtgaactccgtg accgccgccgacaccgccgtgtactactgc huCAN46G13a H, Artificial tggggccagggcaccctggtgaccgtgtcctccg 412 FR4 sequence rehuCAN46G13a K, Artificial gagaacgtgctgacccagtccccctcctccatgtccgcctcc 413 variable sequence gtgggcgaccgcgtgaccatgacctgctccgcctcctcctc region cgtgtcctcctcctacctgcactggtaccagcagaagcccg gcaaggcccccaagcccctgatccaccgcacctccaccct ggcctccggcgtgccctcccgcttctccggctccggctccg gcacctcctactccctgaccatctcctccgtgcagcccgagg acatcgccacctactactgccagcagtggtccggctacccct acaccttcggcggcggcaccaaggtggagatcaagc rehuCAN46G13a K, Artificial tcctccgtgtcctcctcctac 414 CDR1 sequence rehuCAN46G13a K, Artificial cgcacctcc 415 CDR2 sequence rehuCAN46G13a K, Artificial cagcagtggtccggctacccctacacc 416 CDR3 sequence rehuCAN46G13a K, Artificial gagaacgtgctgacccagtccccctcctccatgtccgcctcc 417 FR1 sequence gtgggcgaccgcgtgaccatgacctgctccgcctcc rehuCAN46G13a K, Artificial ctgcactggtaccagcagaagcccggcaaggcccccaag 418 FR2 sequence cccctgatccac rehuCAN46G13a K, Artificial accctggcctccggcgtgccctcccgcttctccggctccgg 419 FR3 sequence ctccggcacctcctactccctgaccatctcctccgtgcagcc cgaggacatcgccacctactactgc rehuCAN46G13a K, Artificial ttcggcggcggcaccaaggtggagatcaagc 420 FR4 sequence rehuCAN46G13a H, Artificial caggtgcagctgcaggagtccggccccggcctggtgaagc 421 variable sequence cctcccagaccctgtccctgacctgcaccgtgaccggctact region ccatcacctccgactccgcctggaactggatccgccagccc cccggcaacggcctggagtggatgggctacatctcctactc cggctccacctcctacaacccctccctgaagtcccgcatctc catcacccgcgacacctccaagaaccagttctccctgaagct gaactccgtgaccgccgccgacaccgccacctactactgc gcccgccgctcccgcgtgtccttctacttcgactactggggc cagggcaccctggtgaccgtgtcctccg rehuCAN46G13a H, Artificial ggctactccatcacctccgactccgcc 422 CDR1 sequence rehuCAN46G13a H, Artificial atctcctactccggctccacc 423 CDR2 sequence rehuCAN46G13a H, Artificial gcccgccgctcccgcgtgtccttctacttcgactac 424 CDR3 sequence rehuCAN46G13a H, Artificial caggtgcagctgcaggagtccggccccggcctggtgaagc 425 FR1 sequence cctcccagaccctgtccctgacctgcaccgtgacc rehuCAN46G13a H, Artificial tggaactggatccgccagccccccggcaacggcctggagt 426 FR2 sequence ggatgggctac rehuCAN46G13a H, Artificial tcctacaacccctccctgaagtcccgcatctccatcacccgc 427 FR3 sequence gacacctccaagaaccagttctccctgaagctgaactccgtg accgccgccgacaccgccacctactactgc rehuCAN46G13a H, Artificial tggggccagggcaccctggtgaccgtgtcctccg 428 FR4 sequence cdrCAN46G19 K, Artificial gacatccagatgacccagtccccctcctccctgtccgcctcc 429 variable sequence gtgggcgaccgcgtgaccatcacctgctccgcctcctcctc region cgtgacctacatgcactggtaccagcagaagcccggcaag gcccccaagctgctgatctacgagacctccaagctggcctc cggcgtgccctcccgcttctccggctccggctccggcaccg actacaccttcaccatctcctccctgcagcccgaggacatcg ccacctactactgcttccagggctccggctaccccttcacctt cggccagggcaccaaggtggagatcaagc cdrCAN46G19 K, Artificial tcctccgtgacctac 430 CDR1 sequence cdrCAN46G19 K, Artificial gagacctcc 431 CDR2 sequence cdrCAN46G19 K, Artificial ttccagggctccggctaccccttcacc 432 CDR3 sequence cdrCAN46G19 K, Artificial gacatccagatgacccagtccccctcctccctgtccgcctcc 433 FR1 sequence gtgggcgaccgcgtgaccatcacctgctccgcctcc cdrCAN46G19 K, Artificial atgcactggtaccagcagaagcccggcaaggcccccaag 434 FR2 sequence ctgctgatctac cdrCAN46G19 K, Artificial aagctggcctccggcgtgccctcccgcttctccggctccgg 435 FR3 sequence ctccggcaccgactacaccttcaccatctcctccctgcagcc cgaggacatcgccacctactactgc cdrCAN46G19 K, Artificial ttcggccagggcaccaaggtggagatcaagc 436 FR4 sequence cdrCAN46G19 H, Artificial Caggtgcagctggtgcagtccggcgccgaggtgaagaag 437 variable sequence cccggcgagtccgtgaaggtgtcctgcaaggcctccggcta region caccttcaccggctactacatccactgggtgcgccaggccc ccggccagggcctggagtggatgggccgcatcttcccctac aacggcgccgcctcctacaaccagaacttcaagggccgcg tgaccatcaccgccgacaagtccacctccaccgcctacatg gagctgtcctccctgcgctccgaggacaccgccgtgtacta ctgcgcccgctggctgcgcgtgtacttcgactactggggcc agggcaccaccgtgaccgtgtcctccg cdrCAN46G19 H, Artificial ggctacaccttcaccggctactac 438 CDR1 sequence cdrCAN46G19 H, Artificial atcttcccctacaacggcgccgcc 439 CDR2 sequence cdrCAN46G19 H, Artificial gcccgctggctgcgcgtgtacttcgactac 440 CDR3 sequence cdrCAN46G19 H, Artificial caggtgcagctggtgcagtccggcgccgaggtgaagaag 441 FR1 sequence cccggcgagtccgtgaaggtgtcctgcaaggcctcc cdrCAN46G19 H, Artificial atccactgggtgcgccaggcccccggccagggcctggagt 442 FR2 sequence ggatgggccgc cdrCAN46G19 H, Artificial tcctacaaccagaacttcaagggccgcgtgaccatcaccgc 443 FR3 sequence cgacaagtccacctccaccgcctacatggagctgtcctccct gcgctccgaggacaccgccgtgtactactgc cdrCAN46G19 H, Artificial tggggccagggcaccaccgtgaccgtgtcctccg 444 FR4 sequence huCAN46G19 K, Artificial gagaacgtgctgacccagtccccctcctccctgtccgcctcc 445 variable sequence gtgggcgaccgcgtgaccatcacctgctccgcctcctcctc region cgtgacctacatgcactggtaccagcagaagcccggcaag gcccccaagctgtggatctacgagacctccaagctggcctc cggcgtgcccggccgcttctccggctccggctccggcaact cctacaccttcaccatctcctccctgcagcccgaggacatcg ccacctactactgcttccagggctccggctaccccttcacctt cggccagggcaccaaggtggagatcaag huCAN46G19 K, Artificial tcctccgtgacctac 446 CDR1 sequence huCAN46G19 K, Artificial gagacctcc 447 CDR2 sequence huCAN46G19 K, Artificial ttccagggctccggctaccccttcacc 448 CDR3 sequence huCAN46G19 K, Artificial gagaacgtgctgacccagtccccctcctccctgtccgcctcc 449 FR1 sequence gtgggcgaccgcgtgaccatcacctgctccgcctcc huCAN46G19 K, Artificial atgcactggtaccagcagaagcccggcaaggcccccaag 450 FR2 sequence ctgtggatctac huCAN46G19 K, Artificial aagctggcctccggcgtgcccggccgcttctccggctccgg 451 FR3 sequence ctccggcaactcctacaccttcaccatctcctccctgcagccc gaggacatcgccacctactactgc huCAN46G19 K, Artificial ttcggccagggcaccaaggtggagatcaag 452 FR4 sequence huCAN46G19 H, Artificial Gaggtgcagctggtgcagtccggcgccgaggtgaagaag 453 variable sequence cccggcgagtccgtgaaggtgtcctgcaaggcctccggcta region ctccttcaccggctactacatccactgggtgaagcaggcccc cggccagggcctggagtgggtgggccgcatcttcccctac aacggcgccgcctcctacaaccagaacttcaagggcaagg ccaccctgaccgtggacaagtcctccaccaccgcctacatg gagctgtcctccctgcgctccgaggacaccgccgtgtacttc tgcgcccgctggctgcgcgtgtacttcgactactggggcca gggcaccaccgtgaccgtgtcctccg huCAN46G19 H, Artificial ggctactccttcaccggctactac 454 CDR1 sequence huCAN46G19 H, Artificial atcttcccctacaacggcgccgcc 455 CDR2 sequence huCAN46G19 H, Artificial gcccgctggctgcgcgtgtacttcgactac 456 CDR3 sequence huCAN46G19 H, Artificial gaggtgcagctggtgcagtccggcgccgaggtgaagaag 457 FR1 sequence cccggcgagtccgtgaaggtgtcctgcaaggcctcc huCAN46G19 H, Artificial atccactgggtgaagcaggcccccggccagggcctggagt 458 FR2 sequence gggtgggccgc huCAN46G19 H, Artificial tcctacaaccagaacttcaagggcaaggccaccctgaccgt 459 FR3 sequence ggacaagtcctccaccaccgcctacatggagctgtcctccct gcgctccgaggacaccgccgtgtacttctgc huCAN46G19 H, Artificial tggggccagggcaccaccgtgaccgtgtcctccg 460 FR4 sequence rehuCAN46G19 K, Artificial gagaacgtgctgacccagtccccctcctccatgtccgcctcc 461 variable sequence gtgggcgaccgcgtgaccatgacctgctccgcctcctcctc region cgtgacctacatgcactggtaccagcagaagcccggcaagt cccccaagctgtggatctacgagacctccaagctggcctcc ggcgtgccctcccgcttctccggctccggctccggcaacga ctactccctgaccatctcctccatgcagcccgaggacgtggc cacctactactgcttccagggctccggctaccccttcaccttc ggccagggcaccaagctggagatcaagc rehuCAN46G19 K, Artificial tcctccgtgacctac 462 CDR1 sequence rehuCAN46G19 K, Artificial gagacctcc 463 CDR2 sequence rehuCAN46G19 K, Artificial ttccagggctccggctaccccttcacc 464 CDR3 sequence rehuCAN46G19 K, Artificial gagaacgtgctgacccagtccccctcctccatgtccgcctcc 465 FR1 sequence gtgggcgaccgcgtgaccatgacctgctccgcctcc rehuCAN46G19 K, Artificial atgcactggtaccagcagaagcccggcaagtcccccaagc 466 FR2 sequence tgtggatctac rehuCAN46G19 K, Artificial aagctggcctccggcgtgccctcccgcttctccggctccgg 467 FR3 sequence ctccggcaacgactactccctgaccatctcctccatgcagcc cgaggacgtggccacctactactgc rehuCAN46G19 K, Artificial ttcggccagggcaccaagctggagatcaagc 468 FR4 sequence rehuCAN46G19 H, Artificial Gaggtgcagctggtgcagtccggcgccgaggtggtgaag 469 variable sequence cccggcgagtccgtgaagatctcctgcaaggcctccggcta region ctccttcaccggctactacatccactgggtgaagcagacccc cggccagtccctggagtgggtgggccgcatcttcccctaca acggcgccgcctcctacaaccagaacttcaagggcaaggc caccctgaccgtggacaagtccaccaccaccgcctacatgg agctgtcctccctgcgctccgaggactccgccgtgtacttct gcgcccgctggctgcgcgtgtacttcgactactggggccag ggcaccaccctgaccgtgtcctccg rehuCAN46G19 H, Artificial ggctactccttcaccggctactac 470 CDR1 sequence rehuCAN46G19 H, Artificial atcttcccctacaacggcgccgcc 471 CDR2 sequence rehuCAN46G19 H, Artificial gcccgctggctgcgcgtgtacttcgactac 472 CDR3 sequence rehuCAN46G19 H, Artificial gaggtgcagctggtgcagtccggcgccgaggtggtgaagc 473 FR1 sequence ccggcgagtccgtgaagatctcctgcaaggcctcc rehuCAN46G19 H, Artificial atccactgggtgaagcagacccccggccagtccctggagt 474 FR2 sequence gggtgggccgc rehuCAN46G19 H, Artificial tcctacaaccagaacttcaagggcaaggccaccctgaccgt 475 FR3 sequence ggacaagtccaccaccaccgcctacatggagctgtcctccc tgcgctccgaggactccgccgtgtacttctgc rehuCAN46G19 H, Artificial tggggccagggcaccaccctgaccgtgtcctccg 476 FR4 sequence cdrCAN46G24 K, Artificial gacatccagatgacccagtccccctcctccctgtccgcctcc 477 variable sequence gtgggcgaccgcgtgaccatcacctgctccgcctcctcctc region cgtgacctacatgcactggtaccagcagaagcccggcaag gcccccaagctgctgatctacgagacctccaagctggcctc cggcgtgccctcccgcttctccggctccggctccggcaccg actacaccttcaccatctcctccctgcagcccgaggacatcg ccacctactactgcttccagggctccggctaccccttcacctt cggccagggcaccaaggtggagatcaagc cdrCAN46G24 K, Artificial tcctccgtgacctac 478 CDR1 sequence cdrCAN46G24 K, Artificial gagacctcc 479 CDR2 sequence cdrCAN46G24 K, Artificial ttccagggctccggctaccccttcacc 480 CDR3 sequence cdrCAN46G24 K, Artificial gacatccagatgacccagtccccctcctccctgtccgcctcc 481 FR1 sequence gtgggcgaccgcgtgaccatcacctgctccgcctcc cdrCAN46G24 K, Artificial atgcactggtaccagcagaagcccggcaaggcccccaag 482 FR2 sequence ctgctgatctac cdrCAN46G24 K, Artificial aagctggcctccggcgtgccctcccgcttctccggctccgg 483 FR3 sequence ctccggcaccgactacaccttcaccatctcctccctgcagcc cgaggacatcgccacctactactgc cdrCAN46G24 K, Artificial ttcggccagggcaccaaggtggagatcaagc 484 FR4 sequence cdrCAN46G24 H, Artificial caggtgcagctggtgcagtccggcgccgaggtgaagaag 485 variable sequence cccggcgagtccgtgaaggtgtcctgcaaggcctccggcta region caccttcaccggctactacatccactgggtgcgccaggccc ccggccagggcctggagtggatgggccgcatcttcccctac aacggcgccgcctcctacaaccagaacttcaagggccgcg tgaccatcaccgccgacaagtccacctccaccgcctacatg gagctgtcctccctgcgctccgaggacaccgccgtgtacta ctgcgcccgctggctgcgcgtgtacttcgactactggggcc agggcaccaccgtgaccgtgtcctccg cdrCAN46G24 H, Artificial ggctacaccttcaccggctactac 486 CDR1 sequence cdrCAN46G24 H, Artificial atcttcccctacaacggcgccgcc 487 CDR2 sequence cdrCAN46G24 H, Artificial gcccgctggctgcgcgtgtacttcgactac 488 CDR3 sequence cdrCAN46G24 H, Artificial caggtgcagctggtgcagtccggcgccgaggtgaagaag 489 FR1 sequence cccggcgagtccgtgaaggtgtcctgcaaggcctcc cdrCAN46G24 H, Artificial atccactgggtgcgccaggcccccggccagggcctggagt 490 FR2 sequence ggatgggccgc cdrCAN46G24 H, Artificial tcctacaaccagaacttcaagggccgcgtgaccatcaccgc 491 FR3 sequence cgacaagtccacctccaccgcctacatggagctgtcctccct gcgctccgaggacaccgccgtgtactactgc cdrCAN46G24 H, Artificial tggggccagggcaccaccgtgaccgtgtcctccg 492 FR4 sequence huCAN46G24 K, Artificial gagatcgtgctgacccagtccccctcctccctgtccacctcc 493 variable sequence gtgggcgaccgcgtgaccatctcctgctccgcctcctcctcc region gtgacctacatgcactggtaccagcagaagcccggcaagg cccccaagctgtggatctacgagacctccaagctggcctcc ggcgtgcccggccgcttctccggctccggctccggcaactc ctacaccttcaccatctcctccctgcagcccgaggacatcgc cacctactactgcttccagggctccggctaccccttcaccttc ggccagggcaccaaggtggagatcaagc huCAN46G24 K, Artificial tcctccgtgacctac 494 CDR1 sequence huCAN46G24 K, Artificial gagacctcc 495 CDR2 sequence huCAN46G24 K, Artificial ttccagggctccggctaccccttcacc 496 CDR3 sequence huCAN46G24 K, Artificial gagatcgtgctgacccagtccccctcctccctgtccacctcc 497 FR1 sequence gtgggcgaccgcgtgaccatctcctgctccgcctcc huCAN46G24 K, Artificial atgcactggtaccagcagaagcccggcaaggcccccaag 498 FR2 sequence ctgtggatctac huCAN46G24 K, Artificial aagctggcctccggcgtgcccggccgcttctccggctccgg 499 FR3 sequence ctccggcaactcctacaccttcaccatctcctccctgcagccc gaggacatcgccacctactactgc huCAN46G24 K, Artificial ttcggccagggcaccaaggtggagatcaagc 500 FR4 sequence huCAN46G24 H, Artificial gaggtgcagctggtgcagtccggcgccgaggtgaagaag 501 variable sequence cccggcgagtccgtgaaggtgtcctgcaaggcctccggcta region ctccttcaccggctactacatccactgggtgaagcaggcccc cggccagggcctggagtgggtgggccgcatcttcccctac aacggcgccgcctcctacaaccagaacttcaagggcaagg ccaccctgaccgtggacaagtcctcctccaccgcctacatg gagctgtcctccctgcgctccgaggacaccgccgtgtacttc tgcgcccgctggctgcgcgtgtacttcgactactggggcca gggcaccaccgtgaccgtgtcctccg huCAN46G24 H, Artificial ggctactccttcaccggctactac 502 CDR1 sequence huCAN46G24 H, Artificial atcttcccctacaacggcgccgcc 503 CDR2 sequence huCAN46G24 H, Artificial gcccgctggctgcgcgtgtacttcgactac 504 CDR3 sequence huCAN46G24 H, Artificial gaggtgcagctggtgcagtccggcgccgaggtgaagaag 505 FR1 sequence cccggcgagtccgtgaaggtgtcctgcaaggcctcc huCAN46G24 H, Artificial atccactgggtgaagcaggcccccggccagggcctggagt 506 FR2 sequence gggtgggccgc huCAN46G24 H, Artificial tcctacaaccagaacttcaagggcaaggccaccctgaccgt 507 FR3 sequence ggacaagtcctcctccaccgcctacatggagctgtcctccct gcgctccgaggacaccgccgtgtacttctgc huCAN46G24 H, Artificial tggggccagggcaccaccgtgaccgtgtcctccg 508 FR4 sequence rehuCAN46G24 K, Artificial gagatcgtgctgacccagtccccctcctccatgtccacctcc 509 variable sequence gtgggcgaccgcgtgaccatgtcctgctccgcctcctcctcc region gtgacctacatgcactggtaccagcagaagcccggcaagtc ccccaagctgtggatctacgagacctccaagctggcctccg gcgtgccctcccgcttctccggctccggctccggcaacgac tactccctgaccatctcctccatgcagcccgaggacgtggcc acctactactgcttccagggctccggctaccccttcaccttcg gccagggcaccaagctggagatcaagc rehuCAN46G24 K, Artificial tcctccgtgacctac 510 CDR1 sequence rehuCAN46G24 K, Artificial gagacctcc 511 CDR2 sequence rehuCAN46G24 K, Artificial ttccagggctccggctaccccttcacc 512 CDR3 sequence rehuCAN46G24 K, Artificial gagatcgtgctgacccagtccccctcctccatgtccacctcc 513 FR1 sequence gtgggcgaccgcgtgaccatgtcctgctccgcctcc rehuCAN46G24 K, Artificial atgcactggtaccagcagaagcccggcaagtcccccaagc 514 FR2 sequence tgtggatctac rehuCAN46G24 K, Artificial aagctggcctccggcgtgccctcccgcttctccggctccgg 515 FR3 sequence ctccggcaacgactactccctgaccatctcctccatgcagcc cgaggacgtggccacctactactgc rehuCAN46G24 K, Artificial ttcggccagggcaccaagctggagatcaagc 516 FR4 sequence rehuCAN46G24 H, Artificial Gaggtgcagctggtgcagtccggcgccgaggtggtgaag 517 variable sequence cccggcgagtccgtgaagatctcctgcaaggcctccggcta region ctccttcaccggctactacatccactgggtgaagcagacccc cggccagtccctggagtgggtgggccgcatcttcccctaca acggcgccgcctcctacaaccagaacttcaagggcaaggc caccctgaccgtggacaagtccacctccaccgcctacatgg agctgtcctccctgcgctccgaggactccgccgtgtacttct gcgcccgctggctgcgcgtgtacttcgactactggggccag ggcaccaccctgaccgtgtcctccg rehuCAN46G24 H, Artificial ggctactccttcaccggctactac 518 CDR1 sequence rehuCAN46G24 H, Artificial atcttcccctacaacggcgccgcc 519 CDR2 sequence rehuCAN46G24 H, Artificial gcccgctggctgcgcgtgtacttcgactac 520 CDR3 sequence rehuCAN46G24 H, Artificial gaggtgcagctggtgcagtccggcgccgaggtggtgaagc 521 FR1 sequence ccggcgagtccgtgaagatctcctgcaaggcctcc rehuCAN46G24 H, Artificial atccactgggtgaagcagacccccggccagtccctggagt 522 FR2 sequence gggtgggccgc rehuCAN46G24 H, Artificial tcctacaaccagaacttcaagggcaaggccaccctgaccgt 523 FR3 sequence ggacaagtccacctccaccgcctacatggagctgtcctccct gcgctccgaggactccgccgtgtacttctgc rehuCAN46G24 H, Artificial tggggccagggcaccaccctgaccgtgtcctccg 524 FR4 sequence cdrCAN46G13a K, Artificial gacattcagatgactcagtctccctcctccctgtctgcttccgt 525 Codon  variable sequence gggggaccgcgtcactattacctgttccgcttcctcctccgtc Optimized region agctcctcttacctgcactggtatcagcagaagccaggaaaa gcccccaagctgctgatctaccggacctccacactggcttct ggcgtgcccagtagattctctggcagtgggtcaggaacaga cttcacttttaccatcagttcactgcagcctgaggatattgcca cttactattgccagcagtggagcggctacccatatacctttgg ccaggggacaaaagtggagatcaaga cdrCAN46G13a K, Artificial tcctccgtcagctcctcttac 526 Codon  CDR1 sequence Optimized cdrCAN46G13a K, Artificial cggacctcc 527 Codon  CDR2 sequence Optimized cdrCAN46G13a K, Artificial cagcagtggagcggctacccatatacc 528 Codon  CDR3 sequence Optimized cdrCAN46G13a K, Artificial gacattcagatgactcagtctccctcctccctgtctgcttccgt 529 Codon  FR1 sequence gggggaccgcgtcactattacctgttccgcttcc Optimized cdrCAN46G13a K, Artificial ctgcactggtatcagcagaagccaggaaaagcccccaagc 530 Codon  FR2 sequence tgctgatctac Optimized cdrCAN46G13a K, Artificial acactggcttctggcgtgcccagtagattctctggcagtggg 531 Codon  FR3 sequence tcaggaacagacttcacttttaccatcagttcactgcagcctg Optimized aggatattgccacttactattgc cdrCAN46G13a K, Artificial tttggccaggggacaaaagtggagatcaaga 532 Codon  FR4 sequence Optimized cdrCAN46G13a H, Artificial caggtgcagctgcaggaatctgggcctggactggtcaaacc 533 Codon  variable sequence ctctcagactctgtctctgacttgtactgtgtccggggggagc Optimized region atcagctccgatagcgcctggaactggatcagacagccccc tgggaagggactggagtggatcgggtacattagttattcagg aagcacctcctacaatccctccctgaaatctagggtcactatg tcagtggacaccagcaagaaccagttctccctgaaagtcaat tctgtgactgccgctgataccgccgtgtactattgcgctcgga gaagtagggtgtcattctactttgactattggggccagggga ccctggtcacagtgtctagtg cdrCAN46G13a H, Artificial ggggggagcatcagctccgatagcgcc 534 Codon  CDR1 sequence Optimized cdrCAN46G13a H, Artificial attagttattcaggaagcacc 535 Codon  CDR2 sequence Optimized cdrCAN46G13a H, Artificial gctcggagaagtagggtgtcattctactttgactat 536 Codon  CDR3 sequence Optimized cdrCAN46G13a H, Artificial caggtgcagctgcaggaatctgggcctggactggtcaaacc 537 Codon  FR1 sequence ctctcagactctgtctctgacttgtactgtgtcc Optimized cdrCAN46G13a H, Artificial tggaactggatcagacagccccctgggaagggactggagt 538 Codon  FR2 sequence ggatcgggtac Optimized cdrCAN46G13a H, Artificial tcctacaatccctccctgaaatctagggtcactatgtcagtgg 539 Codon  FR3 sequence acaccagcaagaaccagttctccctgaaagtcaattctgtga Optimized ctgccgctgataccgccgtgtactattgc cdrCAN46G13a H, Artificial tggggccaggggaccctggtcacagtgtctagtg 540 Codon  FR4 sequence Optimized huCAN46G13a K, Artificial gaaaatgtgctgactcagtccccttccagcctgtccgcaagc 541 Codon  variable sequence gtcggcgacagggtgactatgacctgcagcgcctctagttc Optimized region agtgtccagctcttacctgcactggtatcagcagaagcccgg gaaatctcctaagccactgatccataggacatctactctggct agtggtgtgccttcacggttctctggtagtggctcaggaacat cctacagcctgactatcagttcactgcagccagaggacattg caacctactattgccagcagtggtctggatacccctataccttt ggcggagggacaaaagtggagatcaagc huCAN46G13a K, Artificial agttcagtgtccagctcttac 542 Codon  CDR1 sequence Optimized huCAN46G13a K, Artificial aggacatct 543 Codon  CDR2 sequence Optimized huCAN46G13a K, Artificial cagcagtggtctggatacccctatacc 544 Codon  CDR3 sequence Optimized huCAN46G13a K, Artificial gaaaatgtgctgactcagtccccttccagcctgtccgcaagc 545 Codon  FR1 sequence gtcggcgacagggtgactatgacctgcagcgcctct Optimized huCAN46G13a K, Artificial ctgcactggtatcagcagaagcccgggaaatctcctaagcc 546 Codon  FR2 sequence actgatccat Optimized huCAN46G13a K, Artificial actctggctagtggtgtgccttcacggttctctggtagtggctc 547 Codon  FR3 sequence aggaacatcctacagcctgactatcagttcactgcagccaga Optimized ggacattgcaacctactattgc huCAN46G13a K, Artificial tttggcggagggacaaaagtggagatcaagc 548 Codon  FR4 sequence Optimized huCAN46G13a H, Artificial caggtccagctgcaggaatccgggcctggtctggtgaagc 549 Codon  variable sequence catctcagaccctgagtctgacttgtaccgtgacagggtaca Optimized region gcatcacatctgacagtgcctggaactggattagacagttcc ctggtaacaatctggagtggatgggctacatttcatattccgg aagcacctcttataatcccagtctgaagtcaagaatctccatta cccgcgacacatcaaaaaaccagttttccctgaaggtcaata gcgtgacagctgcagatactgctgtctactattgcgcaaggc ggagccgcgtgtctttctactttgactattggggccagggaa ctctggtcaccgtgtcatccg huCAN46G13a H, Artificial gggtacagcatcacatctgacagtgcc 550 Codon  CDR1 sequence Optimized huCAN46G13a H, Artificial atttcatattccggaagcacc 551 Codon  CDR2 sequence Optimized huCAN46G13a H, Artificial gcaaggcggagccgcgtgtctttctactttgactat 552 Codon  CDR3 sequence Optimized huCAN46G13a H, Artificial caggtccagctgcaggaatccgggcctggtctggtgaagc 553 Codon  FR1 sequence catctcagaccctgagtctgacttgtaccgtgaca Optimized huCAN46G13a H, Artificial tggaactggattagacagttccctggtaacaatctggagtgg 554 Codon  FR2 sequence atgggctac Optimized huCAN46G13a H, Artificial tcttataatcccagtctgaagtcaagaatctccattacccgcg 555 Codon  FR3 sequence acacatcaaaaaaccagttttccctgaaggtcaatagcgtga Optimized cagctgcagatactgctgtctactattgc huCAN46G13a H, Artificial tggggccagggaactctggtcaccgtgtcatccg 556 Codon  FR4 sequence Optimized rehuCAN46G13a K, Artificial gagaacgtcctgacacagtccccttccagcatgtccgcaag 557 Codon  variable sequence cgtcggcgacagggtgactatgacctgctccgcctctagttc Optimized region agtgtccagctcttacctgcactggtatcagcagaagccagg caaagctcccaagcctctgatccataggacatctactctggc aagtggagtgccctcacggttctctggtagtggctcaggaac atcctacagcctgactatcagttcagtgcagcctgaggacatt gctacctactattgccagcagtggagcggctacccatatacc tttggcggagggacaaaagtggagatcaagc rehuCAN46G13a K, Artificial agttcagtgtccagctcttac 558 Codon  CDR1 sequence Optimized rehuCAN46G13a K, Artificial aggacatct 559 Codon  CDR2 sequence Optimized rehuCAN46G13a K, Artificial cagcagtggagcggctacccatatacc 560 Codon  CDR3 sequence Optimized rehuCAN46G13a K, Artificial gagaacgtcctgacacagtccccttccagcatgtccgcaag 561 Codon  FR1 sequence cgtcggcgacagggtgactatgacctgctccgcctct Optimized rehuCAN46G13a K, Artificial ctgcactggtatcagcagaagccaggcaaagctcccaagc 562 Codon  FR2 sequence ctctgatccat Optimized rehuCAN46G13a K, Artificial actctggcaagtggagtgccctcacggttctctggtagtggc 563 Codon  FR3 sequence tcaggaacatcctacagcctgactatcagttcagtgcagcct Optimized gaggacattgctacctactattgc rehuCAN46G13a K, Artificial tttggcggagggacaaaagtggagatcaagc 564 Codon  FR4 sequence Optimized rehuCAN46G13a H, Artificial caggtccagctgcaggaaagcgggcccggtctggtgaagc 565 Codon  variable sequence cttctcagaccctgagtctgacttgtaccgtgacaggatactc Optimized region tatcacatctgacagtgcctggaactggattagacagccacc cggcaatggactggagtggatggggtacatttcatattccgg tagcacatcttataatccaagtctgaagtcaagaatctccatta ctcgcgacacctcaaaaaaccagttctccctgaagctgaata gcgtgactgctgcagatactgctacctactattgcgcaaggc ggagccgcgtgtctttctactttgactattgggggcagggtac actggtcactgtgtcatccg rehuCAN46G13a H, Artificial ggatactctatcacatctgacagtgcc 566 Codon  CDR1 sequence Optimized rehuCAN46G13a H, Artificial atttcatattccggtagcaca 567 Codon  CDR2 sequence Optimized rehuCAN46G13a H, Artificial gcaaggcggagccgcgtgtctttctactttgactat 568 Codon  CDR3 sequence Optimized rehuCAN46G13a H, Artificial caggtccagctgcaggaaagcgggcccggtctggtgaagc 569 Codon  FR1 sequence cttctcagaccctgagtctgacttgtaccgtgaca Optimized rehuCAN46G13a H, Artificial tggaactggattagacagccacccggcaatggactggagtg 711 Codon  FR2 sequence gatggggtac Optimized rehuCAN46G13a H, Artificial tcttataatccaagtctgaagtcaagaatctccattactcgcga  712 Codon  FR3 sequence cacctcaaaaaaccagttctccctgaagctgaatagcgtgac Optimized tgctgcagatactgctacctactattgc rehuCAN46G13a H, Artificial tgggggcagggtacactggtcactgtgtcatccg 713 Codon  FR4 sequence Optimized cdrCAN46G19 K, Artificial gatattcagatgacccagtccccctcctccctgtcagcttccg 714 Codon  variable sequence tcggcgatagagtcaccattacctgttccgctagttcctccgt Optimized region cacatacatgcactggtatcagcagaagccagggaaagcc cccaagctgctgatctacgagactagtaaactggcttcagga gtgccaagcaggttctcaggcagcgggtccggaactgacta tacctttacaatcagctccctgcagcctgaagatattgccacc tactattgcttccagggcagcgggtacccattcacatttggac agggcactaaagtggagatcaagc cdrCAN46G19 K, Artificial tcctccgtcacatac 715 Codon  CDR1 sequence Optimized cdrCAN46G19 K, Artificial gagactagt 716 Codon  CDR2 sequence Optimized cdrCAN46G19 K, Artificial ttccagggcagcgggtacccattcaca 717 Codon  CDR3 sequence Optimized cdrCAN46G19 K, Artificial gatattcagatgacccagtccccctcctccctgtcagcttccg 718 Codon  FR1 sequence tcggcgatagagtcaccattacctgttccgctagt Optimized cdrCAN46G19 K, Artificial atgcactggtatcagcagaagccagggaaagcccccaagc 719 Codon  FR2 sequence tgctgatctac Optimized cdrCAN46G19 K, Artificial aaactggcttcaggagtgccaagcaggttctcaggcagcgg 720 Codon  FR3 sequence gtccggaactgactatacctttacaatcagctccctgcagcct Optimized gaagatattgccacctactattgc cdrCAN46G19 K, Artificial tttggacagggcactaaagtggagatcaagc 570 Codon  FR4 sequence Optimized cdrCAN46G19 H, Artificial caggtgcagctggtccagtccggggccgaggtcaaaaagc 571 Codon  variable sequence ctggggagtccgtcaaagtgtcttgtaaagcatctgggtatac Optimized region atttaccgggtactatatccactgggtgagacaggcacctgg acagggactggagtggatggggaggattttcccatacaacg gagccgccagctataaccagaacttcaagggccgcgtgac aatcactgcagacaaaagtacctcaacagcctacatggagc tgagctccctgcgaagcgaagacacagccgtctactattgc gctcggtggctgagagtgtacttcgattattggggccagggg accacagtcaccgtgtctagtg cdrCAN46G19 H, Artificial gggtatacatttaccgggtactat 572 Codon  CDR1 sequence Optimized cdrCAN46G19 H, Artificial attttcccatacaacggagccgcc 573 Codon  CDR2 sequence Optimized cdrCAN46G19 H, Artificial gctcggtggctgagagtgtacttcgattat 574 Codon  CDR3 sequence Optimized cdrCAN46G19 H, Artificial caggtgcagctggtccagtccggggccgaggtcaaaaagc 575 Codon  FR1 sequence ctggggagtccgtcaaagtgtcttgtaaagcatct Optimized cdrCAN46G19 H, Artificial atccactgggtgagacaggcacctggacagggactggagt 576 Codon  FR2 sequence ggatggggagg Optimized cdrCAN46G19 H, Artificial agctataaccagaacttcaagggccgcgtgacaatcactgc 577 Codon  FR3 sequence agacaaaagtacctcaacagcctacatggagctgagctccc Optimized tgcgaagcgaagacacagccgtctactattgc cdrCAN46G19 H, Artificial tggggccaggggaccacagtcaccgtgtctagtg 578 Codon  FR4 sequence Optimized huCAN46G19 K, Artificial gagaacgtcctgacacagtcaccttccagcctgagcgcctct 579 Codon  variable sequence gtcggtgacagagtgaccatcacatgctctgcttctagttcag Optimized region tgacatacatgcactggtatcagcagaagccaggcaaagca cccaagctgtggatctacgagacttctaagctggcaagtggt gtgccaggacgcttcagtggatcaggatccgggaactcttat acttttaccatctccagcctgcagccagaagatattgctacct actattgcttccagggttccggctaccccttcacatttggaca ggggactaaagtggagatcaaga huCAN46G19 K, Artificial agttcagtgacatac 580 Codon  CDR1 sequence Optimized huCAN46G19 K, Artificial gagacttct 581 Codon  CDR2 sequence Optimized huCAN46G19 K, Artificial ttccagggttccggctaccccttcaca 582 Codon  CDR3 sequence Optimized huCAN46G19 K, Artificial gagaacgtcctgacacagtcaccttccagcctgagcgcctct 583 Codon  FR1 sequence gtcggtgacagagtgaccatcacatgctctgcttct Optimized huCAN46G19 K, Artificial atgcactggtatcagcagaagccaggcaaagcacccaagc 584 Codon  FR2 sequence tgtggatctac Optimized huCAN46G19 K, Artificial aagctggcaagtggtgtgccaggacgcttcagtggatcagg 585 Codon  FR3 sequence atccgggaactcttatacttttaccatctccagcctgcagcca Optimized gaagatattgctacctactattgc huCAN46G19 K, Artificial tttggacaggggactaaagtggagatcaaga 586 Codon  FR4 sequence Optimized huCAN46G19 H, Artificial gaagtccagctggtgcagagcggagcagaggtgaagaaa 587 Codon  variable sequence cctggggaaagcgtcaaagtgtcttgtaaggctagcggata Optimized region ctctttcaccgggtactatatccactgggtcaagcaggcacct ggtcagggactggagtgggtgggtagaattttcccctacaat ggcgctgcaagctataaccagaattttaagggcaaagcaac cctgacagtggacaagagctctaccacagcctacatggagc tgagttcactgcgctctgaagacaccgctgtctatttctgcgc aaggtggctgcgggtgtactttgattattggggacaggggac taccgtcactgtgtccagcg huCAN46G19 H, Artificial ggatactctttcaccgggtactat 588 Codon  CDR1 sequence Optimized huCAN46G19 H, Artificial attttcccctacaatggcgctgca 589 Codon  CDR2 sequence Optimized huCAN46G19 H, Artificial gcaaggtggctgcgggtgtactttgattat 590 Codon  CDR3 sequence Optimized huCAN46G19 H, Artificial gaagtccagctggtgcagagcggagcagaggtgaagaaa 591 Codon  FR1 sequence cctggggaaagcgtcaaagtgtcttgtaaggctagc Optimized huCAN46G19 H, Artificial atccactgggtcaagcaggcacctggtcagggactggagt 592 Codon  FR2 sequence gggtgggtaga Optimized huCAN46G19 H, Artificial agctataaccagaattttaagggcaaagcaaccctgacagtg 593 Codon  FR3 sequence gacaagagctctaccacagcctacatggagctgagttcact Optimized gcgctctgaagacaccgctgtctatttctgc huCAN46G19 H, Artificial tggggacaggggactaccgtcactgtgtccagcg 594 Codon  FR4 sequence Optimized rehuCAN46G19 K, Artificial gagaacgtcctgacacagagtccttccagcatgtcagcctcc 595 Codon  variable sequence gtcggagacagagtgacaatgacttgctctgcttctagttcag Optimized region tgacatacatgcactggtatcagcagaagccagggaaatcc cccaagctgtggatctacgagacttctaagctggcaagtggt gtgccctcacgcttcagcggctctggaagtgggaacgacta tagcctgacaatttccagcatgcagccagaagatgtggcca cttactattgctttcagggttctggctaccccttcacctttggac aggggacaaaactggagatcaaga rehuCAN46G19 K, Artificial agttcagtgacatac 596 Codon  CDR1 sequence Optimized rehuCAN46G19 K, Artificial gagacttct 597 Codon  CDR2 sequence Optimized rehuCAN46G19 K, Artificial tttcagggttctggctaccccttcacc 598 Codon  CDR3 sequence Optimized rehuCAN46G19 K, Artificial gagaacgtcctgacacagagtccttccagcatgtcagcctcc 599 Codon  FR1 sequence gtcggagacagagtgacaatgacttgctctgcttct Optimized rehuCAN46G19 K, Artificial atgcactggtatcagcagaagccagggaaatcccccaagct 600 Codon  FR2 sequence gtggatctac Optimized rehuCAN46G19 K, Artificial aagctggcaagtggtgtgccctcacgcttcagcggctctgg 601 Codon  FR3 sequence aagtgggaacgactatagcctgacaatttccagcatgcagc Optimized cagaagatgtggccacttactattgc rehuCAN46G19 K, Artificial tttggacaggggacaaaactggagatcaaga 602 Codon  FR4 sequence Optimized rehuCAN46G19 H, Artificial gaagtccagctggtgcagtccggagcagaggtggtcaaac 603 Codon  variable sequence ctggggaatctgtgaaaatcagttgtaaggcctcaggatact Optimized region ccttcactgggtactatattcactgggtcaagcagacccctgg tcagagcctggagtgggtgggcagaattttcccctacaatgg agctgcatcttataaccagaattttaagggcaaagcaactctg accgtggacaagagcaccacaactgcctacatggagctga gctctctgcgcagcgaagactctgctgtctatttctgcgcaag gtggctgcgggtgtactttgattattggggtcagggcaccac actgacagtcagttcag rehuCAN46G19 H, Artificial ggatactccttcactgggtactat 604 Codon  CDR1 sequence Optimized rehuCAN46G19 H, Artificial attttcccctacaatggagctgca 605 Codon  CDR2 sequence Optimized rehuCAN46G19 H, Artificial gcaaggtggctgcgggtgtactttgattat 606 Codon  CDR3 sequence Optimized rehuCAN46G19 H, Artificial gaagtccagctggtgcagtccggagcagaggtggtcaaac 607 Codon  FR1 sequence ctggggaatctgtgaaaatcagttgtaaggcctca Optimized rehuCAN46G19 H, Artificial attcactgggtcaagcagacccctggtcagagcctggagtg 608 Codon  FR2 sequence ggtgggcaga Optimized rehuCAN46G19 H, Artificial tcttataaccagaattttaagggcaaagcaactctgaccgtgg 609 Codon  FR3 sequence acaagagcaccacaactgcctacatggagctgagctctctg Optimized cgcagcgaagactctgctgtctatttctgc rehuCAN46G19 H, Artificial tggggtcagggcaccacactgacagtcagttcag 610 Codon  FR4 sequence Optimized cdrCAN46G24 K, Artificial gatattcagatgacccagtccccctcctccctgtcagcttccg 611 Codon  variable sequence tcggcgatagagtcaccattacctgttccgctagttcctccgt Optimized region cacatacatgcactggtatcagcagaagccagggaaagcc cccaagctgctgatctacgagactagtaaactggcttcagga gtgccaagcaggttctcaggcagcgggtccggaactgacta tacctttacaatcagctccctgcagcctgaagatattgccacc tactattgcttccagggcagcgggtacccattcacatttggac agggcactaaagtggagatcaagc cdrCAN46G24 K, Artificial tcctccgtcacatac 612 Codon  CDR1 sequence Optimized cdrCAN46G24 K, Artificial gagactagt 613 Codon  CDR2 sequence Optimized cdrCAN46G24 K, Artificial ttccagggcagcgggtacccattcaca 614 Codon  CDR3 sequence Optimized cdrCAN46G24 K, Artificial gatattcagatgacccagtccccctcctccctgtcagcttccg 615 Codon  FR1 sequence tcggcgatagagtcaccattacctgttccgctagt Optimized cdrCAN46G24 K, Artificial atgcactggtatcagcagaagccagggaaagcccccaagc 616 Codon  FR2 sequence tgctgatctac Optimized cdrCAN46G24 K, Artificial aaactggcttcaggagtgccaagcaggttctcaggcagcgg 617 Codon  FR3 sequence gtccggaactgactatacctttacaatcagctccctgcagcct Optimized gaagatattgccacctactattgc cdrCAN46G24 K, Artificial tttggacagggcactaaagtggagatcaagc 618 Codon  FR4 sequence Optimized cdrCAN46G24 H, Artificial caggtgcagctggtccagtccggggccgaggtcaaaaagc 619 Codon  variable sequence ctggggagtccgtcaaagtgtcttgtaaagcatctgggtatac Optimized region atttaccgggtactatatccactgggtgagacaggcacctgg acagggactggagtggatggggaggattttcccatacaacg gagccgccagctataaccagaacttcaagggccgcgtgac aatcactgcagacaaaagtacctcaacagcctacatggagc tgagctccctgcgaagcgaagacacagccgtctactattgc gctcggtggctgagagtgtacttcgattattggggccagggg accacagtcaccgtgtctagtg cdrCAN46G24 H, Artificial gggtatacatttaccgggtactat 620 Codon  CDR1 sequence Optimized cdrCAN46G24 H, Artificial attttcccatacaacggagccgcc 621 Codon  CDR2 sequence Optimized cdrCAN46G24 H, Artificial gctcggtggctgagagtgtacttcgattat 622 Codon  CDR3 sequence Optimized cdrCAN46G24 H, Artificial caggtgcagctggtccagtccggggccgaggtcaaaaagc 623 Codon  FR1 sequence ctggggagtccgtcaaagtgtcttgtaaagcatct Optimized cdrCAN46G24 H, Artificial atccactgggtgagacaggcacctggacagggactggagt 624 Codon  FR2 sequence ggatggggagg Optimized cdrCAN46G24 H, Artificial agctataaccagaacttcaagggccgcgtgacaatcactgc 625 Codon  FR3 sequence agacaaaagtacctcaacagcctacatggagctgagctccc Optimized tgcgaagcgaagacacagccgtctactattgc cdrCAN46G24 H, Artificial tggggccaggggaccacagtcaccgtgtctagtg 626 Codon  FR4 sequence Optimized huCAN46G24 K, Artificial gagatcgtcctgactcagtccccttccagcctgtctaccagtg 627 Codon  variable sequence tcggtgacagagtgacaatctcatgctccgcttctagttcagt Optimized region gacatacatgcactggtatcagcagaagccaggcaaagcc cccaagctgtggatctacgagacttccaagctggctagcggt gtgccaggacgcttcagcggatctggaagtgggaactcttat accttcaccatctccagcctgcagccagaagatattgctacct actattgcttccagggttccggctaccccttcacctttggaca ggggacaaaagtggagatcaaga huCAN46G24 K, Artificial agttcagtgacatac 628 Codon  CDR1 sequence Optimized huCAN46G24 K, Artificial gagacttcc 629 Codon  CDR2 sequence Optimized huCAN46G24 K, Artificial ttccagggttccggctaccccttcacc 630 Codon  CDR3 sequence Optimized huCAN46G24 K, Artificial gagatcgtcctgactcagtccccttccagcctgtctaccagtg 631 Codon  FR1 sequence tcggtgacagagtgacaatctcatgctccgcttct Optimized huCAN46G24 K, Artificial atgcactggtatcagcagaagccaggcaaagcccccaagc 632 Codon  FR2 sequence tgtggatctac Optimized huCAN46G24 K, Artificial aagctggctagcggtgtgccaggacgcttcagcggatctgg 633 Codon  FR3 sequence aagtgggaactcttataccttcaccatctccagcctgcagcca Optimized gaagatattgctacctactattgc huCAN46G24 K, Artificial tttggacaggggacaaaagtggagatcaaga 634 Codon  FR4 sequence Optimized huCAN46G24 H, Artificial gaagtccagctggtgcagagcggagcagaggtgaagaaa 635 Codon  variable sequence cctggggaatcagtcaaagtgtcctgtaaggcatcaggatac Optimized region tccttcaccgggtactatatccactgggtcaagcaggcacct ggtcagggactggagtgggtgggtagaattttcccctacaat ggcgctgcaagctataaccagaattttaagggcaaagcaac tctgaccgtggacaagagctctagtacagcctacatggagct gtcatccctgcgctctgaagacactgctgtctatttctgcgca aggtggctgcgggtgtactttgattattggggacaggggacc acagtcacagtgagctctg huCAN46G24 H, Artificial ggatactccttcaccgggtactat 636 Codon  CDR1 sequence Optimized huCAN46G24 H, Artificial attttcccctacaatggcgctgca 637 Codon  CDR2 sequence Optimized huCAN46G24 H, Artificial gcaaggtggctgcgggtgtactttgattat 638 Codon  CDR3 sequence Optimized huCAN46G24 H, Artificial gaagtccagctggtgcagagcggagcagaggtgaagaaa 639 Codon  FR1 sequence cctggggaatcagtcaaagtgtcctgtaaggcatca Optimized huCAN46G24 H, Artificial atccactgggtcaagcaggcacctggtcagggactggagt 640 Codon  FR2 sequence gggtgggtaga Optimized huCAN46G24 H, Artificial agctataaccagaattttaagggcaaagcaactctgaccgtg 641 Codon  FR3 sequence gacaagagctctagtacagcctacatggagctgtcatccctg Optimized cgctctgaagacactgctgtctatttctgc huCAN46G24 H, Artificial tggggacaggggaccacagtcacagtgagctctg 642 Codon  FR4 sequence Optimized rehuCAN46G24 K, Artificial gagatcgtgctgactcagtcaccctccagcatgtcaacctcc 643 Codon  variable sequence gtcggagacagagtgacaatgagctgctctgcctctagttca Optimized region gtgacctacatgcactggtatcagcagaagccagggaaaa gccccaagctgtggatctacgagacaagcaagctggcttct ggtgtgcccagtcgcttcagtggctcaggatccgggaacga ctattccctgaccatttccagcatgcagccagaagatgtggc aacatactattgctttcagggtagcggctaccccttcacctttg gacaggggacaaaactggagatcaaga rehuCAN46G24 K, Artificial agttcagtgacctac 644 Codon  CDR1 sequence Optimized rehuCAN46G24 K, Artificial gagacaagc 645 Codon  CDR2 sequence Optimized rehuCAN46G24 K, Artificial tttcagggtagcggctaccccttcacc 646 Codon  CDR3 sequence Optimized rehuCAN46G24 K, Artificial gagatcgtgctgactcagtcaccctccagcatgtcaacctcc 647 Codon  FR1 sequence gtcggagacagagtgacaatgagctgctctgcctct Optimized rehuCAN46G24 K, Artificial atgcactggtatcagcagaagccagggaaaagccccaagc 648 Codon  FR2 sequence tgtggatctac Optimized rehuCAN46G24 K, Artificial aagctggcttctggtgtgcccagtcgcttcagtggctcagga 649 Codon  FR3 sequence tccgggaacgactattccctgaccatttccagcatgcagcca Optimized gaagatgtggcaacatactattgc rehuCAN46G24 K, Artificial tttggacaggggacaaaactggagatcaaga 650 Codon  FR4 sequence Optimized rehuCAN46G24 H, Artificial gaagtccagctggtgcagtccggagcagaggtggtcaaac 651 Codon  variable sequence ctggggaaagcgtgaaaatctcttgtaaggctagtggatact Optimized region cattcacagggtactatattcactgggtcaagcagactccag gccagtctctggagtgggtgggcagaattttcccctacaatg gagctgcatcctataaccagaattttaagggcaaagcaaccc tgacagtggacaagagcacttctaccgcctacatggagctg agctctctgcgctccgaagacagcgctgtctatttctgcgcaa ggtggctgcgggtgtactttgattattggggtcagggcacca cactgacagtcagttcag rehuCAN46G24 H, Artificial ggatactcattcacagggtactat 652 Codon  CDR1 sequence Optimized rehuCAN46G24 H, Artificial attttcccctacaatggagctgca 653 Codon  CDR2 sequence Optimized rehuCAN46G24 H, Artificial gcaaggtggctgcgggtgtactttgattat 654 Codon  CDR3 sequence Optimized rehuCAN46G24 H, Artificial gaagtccagctggtgcagtccggagcagaggtggtcaaac 655 Codon  FR1 sequence ctggggaaagcgtgaaaatctcttgtaaggctagt Optimized rehuCAN46G24 H, Artificial attcactgggtcaagcagactccaggccagtctctggagtg 656 Codon  FR2 sequence ggtgggcaga Optimized rehuCAN46G24 H, Artificial tcctataaccagaattttaagggcaaagcaaccctgacagtg 657 Codon  FR3 sequence gacaagagcacttctaccgcctacatggagctgagctctctg Optimized cgctccgaagacagcgctgtctatttctgc rehuCAN46G24 H, Artificial tggggtcagggcaccacactgacagtcagttcag 658 Codon  FR4 sequence Optimized cdrCAN46G4 K, Artificial gaaattgtcctgacccagtcccctgctaccctgtccctgtccc 659 Codon  variable sequence ccggagaaagagcaaccctgtcctgttcagcttcctcatctgt Optimized region gtcttacatgcactggtatcagcagaagccagggcaggcac ccaggctgctgatctacgagactagtaaactggcattcggaa ttcccgcacgcttttcaggcagcgggtccggaaccgacttca ccctgacaatcagctccctggagcctgaagatttcgccgtgt actattgctttcagggcagcgggtatccattcacatttggaca gggcactcggctggagatcaaga cdrCAN46G4 K, Artificial tcatctgtgtcttac 660 Codon  CDR1 sequence Optimized cdrCAN46G4 K, Artificial gagactagt 661 Codon  CDR2 sequence Optimized cdrCAN46G4 K, Artificial tttcagggcagcgggtatccattcaca 662 Codon  CDR3 sequence Optimized cdrCAN46G4 K, Artificial gaaattgtcctgacccagtcccctgctaccctgtccctgtccc 663 Codon  FR1 sequence ccggagaaagagcaaccctgtcctgttcagcttcc Optimized cdrCAN46G4 K, Artificial atgcactggtatcagcagaagccagggcaggcacccaggc 664 Codon  FR2 sequence tgctgatctac Optimized cdrCAN46G4 K, Artificial aaactggcattcggaattcccgcacgcttttcaggcagcggg 665 Codon  FR3 sequence tccggaaccgacttcaccctgacaatcagctccctggagcct Optimized gaagatttcgccgtgtactattgc cdrCAN46G4 K, Artificial tttggacagggcactcggctggagatcaaga 666 Codon  FR4 sequence Optimized cdrCAN46G4 H, Artificial caggtccagctggtccagtctggggctgaggtcaaaaaacc 667 Codon  variable sequence cggctcttccgtcaaagtctcctgcaaagcatctggctataca Optimized region tttaccgggtactatatgcactgggtgagacaggcacctggg cagggactggagtggatcgggaggattttcccatacaacgg agccgccagctataaccagaacttcaaggacaaagccacta tcaccgctgatgaaagtacaaatactgcctacatggagctga gctccctgaggtctgaagacactgcagtctactattgcgccc ggtggctgagagtgtacttcgattattggggccaggggaca ctggtcaccgtgagcagtg cdrCAN46G4 H, Artificial ggctatacatttaccgggtactat 668 Codon  CDR1 sequence Optimized cdrCAN46G4 H, Artificial attttcccatacaacggagccgcc 669 Codon  CDR2 sequence Optimized cdrCAN46G4 H, Artificial gcccggtggctgagagtgtacttcgattat 670 Codon  CDR3 sequence Optimized cdrCAN46G4 H, Artificial Caggtccagctggtccagtctggggctgaggtcaaaaaac 671 Codon  FR1 sequence ccggctcttccgtcaaagtctcctgcaaagcatct Optimized cdrCAN46G4 H, Artificial atgcactgggtgagacaggcacctgggcagggactggagt 672 Codon  FR2 sequence ggatcgggagg Optimized cdrCAN46G4 H, Artificial agctataaccagaacttcaaggacaaagccactatcaccgct 673 Codon  FR3 sequence gatgaaagtacaaatactgcctacatggagctgagctccctg Optimized aggtctgaagacactgcagtctactattgc cdrCAN46G4 H, Artificial tggggccaggggacactggtcaccgtgagcagtg 674 Codon  FR4 sequence Optimized huCAN46G4 K, Artificial gagaaggtcctgacacagtcacccgctaccctgtccctgag 675 Codon  variable sequence ccctggcgagagagccactatgacctgctcagcttccagct Optimized region ctgtgtcctacatgcactggtatcagcagaagccaggaacct ctcccaaactgtggatctacgaaaccagtaagctggctttcg gggtgccagcacgcttttctggcagtggatcagggaactcct atagcctgaccattagttcactggaaccagaagacttcgctgt gtactattgctttcagggtagcggctaccccttcacctttggac aggggacaagactggagatcaagc huCAN46G4 K, Artificial agctctgtgtcctac 676 Codon  CDR1 sequence Optimized huCAN46G4 K, Artificial gaaaccagt 677 Codon  CDR2 sequence Optimized huCAN46G4 K, Artificial tttcagggtagcggctaccccttcacc 678 Codon  CDR3 sequence Optimized huCAN46G4 K, Artificial gagaaggtcctgacacagtcacccgctaccctgtccctgag 679 Codon  FR1 sequence ccctggcgagagagccactatgacctgctcagcttcc Optimized huCAN46G4 K, Artificial atgcactggtatcagcagaagccaggaacctctcccaaact 680 Codon  FR2 sequence gtggatctac Optimized huCAN46G4 K, Artificial aagctggctttcggggtgccagcacgcttttctggcagtgga 681 Codon  FR3 sequence tcagggaactcctatagcctgaccattagttcactggaacca Optimized gaagacttcgctgtgtactattgc huCAN46G4 K, Artificial tttggacaggggacaagactggagatcaagc 682 Codon  FR4 sequence Optimized huCAN46G4 H, Artificial gaagtgcagctgctgcagtccggagctgaggtcaagaaac 683 Codon  variable sequence ccgggtcatccgtgaagattagctgtaaagcatctgattaca Optimized region gttttaccggctactatatgcactgggtgaagcaggcacctg gtcagggactggagtggatcggtagaattttcccctacaatg gcgctgcatcctataaccagaattttaaggacaaagctaccct gacagtggataagagctctagtaccgcatatatggagctgca ttcactgcgctccgaagacacagccgtctactattgcactag gtggctgcgggtgtacttcgattattggggacaggggaccct ggtcacagtgtcatccg huCAN46G4 H, Artificial gattacagttttaccggctactat 684 Codon  CDR1 sequence Optimized huCAN46G4 H, Artificial attttcccctacaatggcgctgca 685 Codon  CDR2 sequence Optimized huCAN46G4 H, Artificial actaggtggctgcgggtgtacttcgattat 686 Codon  CDR3 sequence Optimized huCAN46G4 H, Artificial gaagtgcagctgctgcagtccggagctgaggtcaagaaac 687 Codon  FR1 sequence ccgggtcatccgtgaagattagctgtaaagcatct Optimized huCAN46G4 H, Artificial atgcactgggtgaagcaggcacctggtcagggactggagt 688 Codon  FR2 sequence ggatcggtaga Optimized huCAN46G4 H, Artificial tcctataaccagaattttaaggacaaagctaccctgacagtg 689 Codon  FR3 sequence gataagagctctagtaccgcatatatggagctgcattcactgc Optimized gctccgaagacacagccgtctactattgc huCAN46G4 H, Artificial tggggacaggggaccctggtcacagtgtcatccg 690 Codon  FR4 sequence Optimized rehuCAN46G4 K, Artificial gaaaaggtcctgactcagtcccccgctactctgtcagcatcc 691 Codon  variable sequence cctggcgagagagtcaccatgagctgctctgcctccagctct Optimized region gtgtcttacatgcactggtatcagcagaagcctggtcagagt cccaaactgtggatctacgaaacttcaaagctggcattcggc gtgccagcccgctttagtggctcaggatccgggaccgacta ttccctgacaattagttcaatggagccagaagatttcgctacat actattgctttcagggtagcggctaccccttcacttttggacag gggaccagactggagatcaagc rehuCAN46G4 K, Artificial agctctgtgtcttac 692 Codon  CDR1 sequence Optimized rehuCAN46G4 K, Artificial gaaacttca 693 Codon  CDR2 sequence Optimized rehuCAN46G4 K, Artificial tttcagggtagcggctaccccttcact 694 Codon  CDR3 sequence Optimized rehuCAN46G4 K, Artificial gaaaaggtcctgactcagtcccccgctactctgtcagcatcc 695 Codon  FR1 sequence cctggcgagagagtcaccatgagctgctctgcctcc Optimized rehuCAN46G4 K, Artificial atgcactggtatcagcagaagcctggtcagagtcccaaact 696 Codon  FR2 sequence gtggatctac Optimized rehuCAN46G4 K, Artificial aagctggcattcggcgtgccagcccgctttagtggctcagg 697 Codon  FR3 sequence atccgggaccgactattccctgacaattagttcaatggagcc Optimized agaagatttcgctacatactattgc rehuCAN46G4 K, Artificial tttggacaggggaccagactggagatcaagc 698 Codon  FR4 sequence Optimized rehuCAN46G4 H, Artificial gaagtgcagctgctgcagtccggtgcagaggtggtcaagc 699 Codon  variable sequence caggatcatccgtgaagattagctgtaaagctagcggttact Optimized region cttttaccggctactatatgcactgggtgaagcaggcacctg gtcagggcctggagtggatcggaagaattttcccctacaac ggggctgcatcttataaccagaattttaaggacaaagccaca ctgactgctgataagtccaccaatacagcatatatggagctg agctctctgcgcagtgaagactcagccgtctactattgcacc aggtggctgcgggtgtacttcgattattggggacaggggac cctggtcacagtgagttcag rehuCAN46G4 H, Artificial ggttactcttttaccggctactat 700 Codon  CDR1 sequence Optimized rehuCAN46G4 H, Artificial attttcccctacaacggggctgca 701 Codon  CDR2 sequence Optimized rehuCAN46G4 H, Artificial accaggtggctgcgggtgtacttcgattat 702 Codon  CDR3 sequence Optimized rehuCAN46G4 H, Artificial Gaagtgcagctgctgcagtccggtgcagaggtggtcaagc 703 Codon  FR1 sequence caggatcatccgtgaagattagctgtaaagctagc Optimized rehuCAN46G4 H, Artificial atgcactgggtgaagcaggcacctggtcagggcctggagt 704 Codon  FR2 sequence ggatcggaaga Optimized rehuCAN46G4 H, Artificial tcttataaccagaattttaaggacaaagccacactgactgctg 705 Codon  FR3 sequence ataagtccaccaatacagcatatatggagctgagctctctgc Optimized gcagtgaagactcagccgtctactattgc rehuCAN46G4 H, Artificial tggggacaggggaccctggtcacagtgagttcag 706 Codon  FR4 sequence Optimized Chimeric K, Artificial gagaatgtcctgactcagtcccctgctattatggccgcttccc 707 CAN46G13a variable sequence tggggcagaaagtgactatgacctgttccgcttcctcttccgt region cagctcctcttacctgcactggtatcagcagaagtctggcgct agtccaaaacccctgatccatcgaaccagcacactggcttcc ggagtgccagcaagattctctggcagtgggtcaggaacaa gctactccctgactattagttcagtcgaggcagaagacgatg ccacctactattgccagcagtggtctgggtacccttataccttt ggcgggggaacaaagctggagatcaaa K, Artificial ENVLTQSPAIMAASLGQKVTMTCSASS 708 variable sequence SVSSSYLHWYQQKSGASPKPLIHRTST region LASGVPARFSGSGSGTSYSLTISSVEAE DDATYYCQQWSGYPYTFGGGTKLEIK H, Artificial gacgtgcagctgcaggaatctgggcctgggctggtgaaac 709 variable sequence ctagtcagtctctgtctctgacctgtaccgtgaccggatactc region aatcacctccgattctgcctggaactggatcaggcagttccct ggcaacaatctggagtggatgggatacattagttattcaggc agcacatcctacaatccatccctgaagtctaggatcagtatta cccgcgacacaagtaaaaaccagttctttctgcagctgaattc agtgaccacagaagataccgctacatactattgcgcacgga gatcacgggtgagcttctactttgactattgggggcagggaa ctaccctgactgtcagctcc H, Artificial DVQLQESGPGLVKPSQSLSLTCTVTGY 710 variable sequence SITSDSAWNWIRQFPGNNLEWMGYISY region SGSTSYNPSLKSRISITRDTSKNQFFLQL NSVTTEDTATYYCARRSRVSFYFDYW GQGTTLTVSS

In Table 1, for amino acid sequences the CDRs are underlined and for nucleotide sequences the CDRs are IMGT numbering. H: heavy chain; K: kappa chain. The CDR regions can be identified using Kabat, IMGT, Honnegger, and Chothia and can vary accordingly.

As used herein, “homologous to” means “at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous” to a defined sequence, amino acid or nucleotide. If a range if specified, e.g., about 80% to about 100%, then the term homologous as used in therein refers to that range particularly.

The antibodies or antigen-binding portions can comprise: (1) a heavy chain variable region comprising an amino acid sequence homologous to a heavy chain variable region amino acid sequence of the antibody produced by clone CAN33G1 (SEQ ID NO. 93), CAN46G4 (SEQ ID NO: 11), CAN46G13 (SEQ ID NO: 27), CAN46G13a (SEQ ID NO: 43), CAN46G19 (SEQ ID NO: 59), or CAN46G24 (SEQ ID NO: 75); and/or, (2) a light chain variable region comprising an amino acid sequence homologous to a light chain variable region amino acid sequence of the antibodies produced by clones CAN33G1 (SEQ ID NO. 85), CAN46G4 (SEQ ID NO: 3), CAN46G13 (SEQ ID NO: 19), CAN46G13a (SEQ ID NO: 35), CAN46G19 (SEQ ID NO: 51), or CAN46G24 (SEQ ID NO: 67). The antibodies or antigen-binding portions can comprise any combination of the amino acid sequences set forth for the heavy chain variable region (1) and light chain variable region (2) listed in the preceeding paragraph.

The antibodies or antigen-binding portions can specifically bind to an epitope that overlaps with, is antigenically similar to, or is homologous to, either at the amino acid or nucleotide sequence level, an epitope bound by an antibody produced by clones, including, but not limited to, humanized antibodies derived from these clones, CAN46G4, CAN46G13, CAN46G13a, CAN46G19, CAN46G24 or CAN33G1 and can compete for binding to toxin B with an antibody produced by clones CAN46G4, CAN46G13, CAN46G13a, CAN46G19, CAN46G24 or CAN33G1.

In one embodiment, the antibodies or antigen-binding portions of the present invention comprises: (1) a heavy chain variable region comprising one, two, three or more complementarity determining regions (CDRs) that are homologous to one, two, three or more CDRs of the antibodies produced by clones, CAN33G1 (SEQ ID NOs: 94, 95 and/or 96), CAN46G4 (SEQ ID NOs: 12, 13 and/or 14), CAN46G13 (SEQ ID NOs: 28, 29 and/or 30), CAN46G13a (SEQ ID NOs: 44, 45 and/or 46), CAN46G19 (SEQ ID NOs: 60, 61 and/or 62), or CAN46G24 (SEQ ID NOs: 76, 77 and/or 78); or, (2) a light chain variable region comprising one, two, three or more CDRs that are homologous to one, two, three or more CDRs of the antibodies produced by clones, CAN33G1 (SEQ ID NOs: 86, 87 and/or 88), CAN46G4 (SEQ ID NOs: 4, 5 and/or 6), CAN46G13 (SEQ ID NOs: 20, 21 and/or 22), CAN46G13a (SEQ ID NOs: 36, 37 and/or 38), CAN46G19 (SEQ ID NOs: 52, 53 and/or 54), or CAN46G24 (SEQ ID NOs: 68, 69 and/or 70). The antibodies or antigen-binding portions can comprise any combination of the amino acid sequences set forth for the heavy chain variable region (1) and light chain variable region (2) listed in the preceeding paragraph.

In another embodiment, the antibodies or antigen-binding portions can comprise: (i) a heavy chain variable region of the antibodies or antigen-binding portion comprising one, two, three or more complementarity determining regions (CDRs) that are homologous to one, two, three or more CDRs of the antibodies produced by clones cdrCAN46G13a (SEQ ID NOs:110, 111 and/or 112), huCAN46G13a (SEQ ID NOs: 126, 127 and/or 128), rehuCAN46G13a (SEQ ID NOs: 142, 143 and/or 144), cdrCAN46G19 (SEQ ID NOs: 158, 159 and/or 160), huCAN46G19 (SEQ ID NOs: 174, 175 and/or 176), rehuCAN46G19 (SEQ ID NOs: 190, 191 and/or 192), cdrCAN46G24 (SEQ ID NOs: 206, 207 and/or 208), huCAN46G24 (SEQ ID NOs: 222, 223 and/or 224) or rehuCAN46G24 (SEQ ID NOs: 238, 239 and/or 240); or (ii) a light chain variable region of the antibodies or antigen-binding portion comprising one, two, three or more CDRs that are homologous to one, two, three or more CDRs of a light chain variable region of the antibodies produced by the clones cdrCAN46G13a (SEQ ID NOs: 102, 103 and/or 104), huCAN46G13a (SEQ ID NOs: 118, 119 and/or 120), rehuCAN46G13a (SEQ ID NOs: 134, 135 and/or 136), cdrCAN46G19 (SEQ ID NOs: 150, 151 and/or 152), huCAN46G19 (SEQ ID NOs: 166, 167 and/or 168), rehuCAN46G19 (SEQ ID NOs: 182, 183 and/or 184), cdrCAN46G24 (SEQ ID NOs: 198, 199 and/or 200), huCAN46G24 (SEQ ID NOs: 214, 215 and/or 216) or rehuCAN46G24 (SEQ ID NOs: 230, 231 and/or 232). The antibodies or antigen-binding portions can comprise any combination of the amino acid sequences set forth for the CDRs for heavy chain variable region and light chain variable region listed in the preceeding paragraph.

In a third embodiment, the antibodies or antigen-binding portion comprise: (i) a heavy chain variable region of the antibodies or antigen-binding portion which comprises one, two, three or more complementarity determining regions (CDRs) that are homologous to one, two, three or more CDRs of the antibodies produced by clones, cdrCAN46G13a (SEQ ID NOs:110, 111 and/or 112), huCAN46G13a (SEQ ID NOs: 126, 127 and/or 128), rehuCAN46G13a (SEQ ID NOs: 142, 143 and/or 144), cdrCAN46G19 (SEQ ID NOs: 158, 159 and/or 160), huCAN46G19 (SEQ ID NOs: 174, 175 and/or 176), rehuCAN46G19 (SEQ ID NOs: 190, 191 and/or 192), cdrCAN46G24 (SEQ ID NOs: 206, 207 and/or 208), huCAN46G24 (SEQ ID NOs: 222, 223 and/or 224) or rehuCAN46G24 (SEQ ID NOs: 238, 239 and/or 240); or (ii) a light chain variable region of the antibodies or antigen-binding portion comprising one, two, three or more CDRs that are homologous to one, two, three or more CDRs of a light chain variable region of the antibody produced by clones, cdrCAN46G13a (SEQ ID NOs: 102, 103 and/or 104), huCAN46G13a (SEQ ID NOs: 118, 119 and/or 120), rehuCAN46G13a (SEQ ID NOs: 134, 135 and/or 136), cdrCAN46G19 (SEQ ID NOs: 150, 151 and/or 152), huCAN46G19 (SEQ ID NOs: 166, 167 and/or 168), rehuCAN46G19 (SEQ ID NOs: 182, 183 and/or 184), cdrCAN46G24 (SEQ ID NOs: 198, 199 and/or 200), huCAN46G24 (SEQ ID NOs: 214, 215 and/or 216) or rehuCAN46G24 (SEQ ID NOs: 230, 231 and/or 232).

In certain embodiments, the antibody or antigen-binding portion comprises a light chain variable region and heavy chain variable region homologous to a light chain variable region and heavy chain variable region of the antibodies produced by clones cdrCAN46G13a (SEQ ID NOs: 101 and 109, light chain variable region—heavy chain variable region, respectively), huCAN46G13a (SEQ ID NOs: 117 and 125, light chain variable region—heavy chain variable region, respectively), rehuCAN46G13a (SEQ ID NOs: 133 and 141, light chain variable region—heavy chain variable region, respectively), chimeric CAN46G13a (SEQ ID NOs: 708 and 710, light chain variable region—heavy chain variable region, respectively), cdrCAN46G19 (SEQ ID NOs: 149 and 157, light chain variable region—heavy chain variable region, respectively), huCAN46G19 (SEQ ID NOs: 165 and 173, light chain variable region—heavy chain variable region, light chain variable region—heavy chain variable region, respectively), rehuCAN46G19 (SEQ ID NOs: 181 and 189, respectively), cdrCAN46G24 (SEQ ID NOs: 197 and 205, respectively), huCAN46G24 (SEQ ID NOs: 213 and 221, light chain variable region—heavy chain variable region, respectively) or rehuCAN46G24 (SEQ ID NOs: 229 and 237, light chain variable region—heavy chain variable region, respectively).

The antibody or antigen-binding portion can comprise: (i) a light chain variable region homologous to a light chain variable region as set forth in SEQ ID NOs: 101, 117, 133, 708, 149, 165 or 181, 197, 213 or 229; or (ii) a heavy chain variable region homologous to a heavy chain variable region as set forth in SEQ ID NOs: 109, 125, 141, 710, 157, 173, 189, 205, 221 or 237. In certain embodiments, the antibody or antigen-binding portions comprise both heavy chain variable regions (i) and light chain variable regions (ii).

The antibodies or antigen-binding portion can comprise: (i) a heavy chain variable region encoded by a nucleic acid sequence homologous to a nucleic acid sequence as set forth in SEQ ID NOs: 667, 683, 699, 389, 405, 421, 533, 549, 565, 709, 437, 453, 469, 571, 587, 603, 485, 501, 517, 619, 635, or 651; (ii) a light chain variable region encoded by a nucleic acid sequence homologous to a nucleic acid sequence as set forth in SEQ ID NOs: 659, 675, 691, 381, 397, 413, 525, 541, 557, 707, 429, 445, 461, 557, 579, 595, 477, 493, 509, 611, 627, or 643; and/or, (iii) both a heavy chain variable region encoded by a nucleic acid sequence homologous to a nucleic acid sequence as set forth in SEQ ID NOs: 667, 683, 699, 389, 405, 421, 533, 549, 565, 709, 437, 453, 469, 571, 587, 603, 485, 501, 517, 619, 635, or 651, and a light chain variable region encoded by a nucleic acid sequence homologous to a nucleic acid sequence as set forth in SEQ ID NOs: 659, 675, 691, 381, 397, 413, 525, 541, 557, 707, 429, 445, 461, 557, 579, 595, 477, 493, 509, 611, 627, or 643.

The antibodies or antigen-binding portion can comprise one, two, three or more complementarity determining regions (CDRs) encoded by nucleic acid sequences that are homologous to one, two, three or more CDRs encoded by the nucleic acid sequence of: (i) the cdrs of the heavy chain variable region as set forth in, cdrCAN46G4 (SEQ ID NOs: 668, 669 and/or 670), huCAN46G4 (SEQ ID NOs: 684, 685 and/or 686), rehuCAN46G4 (SEQ ID NOs: 700, 701 and/or 702), cdrCAN46G13a (SEQ ID NOs: (a) 390, 391 and/or 392, or (b) 534, 535 and/or 536), huCAN46G13a (SEQ ID NOs: (a) 406, 407 and/or 408, or (b) 550, 551 and/or 552), rehuCAN46G13a (SEQ ID NOs: (a) 422, 423 and/or 424, or (b) 566, 567 and/or 568), cdrCAN46G19 (SEQ ID NOs: (a) 438, 439 and 440, or (b) 572, 573 and/or 574), huCAN46G19 (SEQ ID NOs: (a) 454, 455 and/or 456, or (b) 588, 589 and/or 590), rehuCAN46G19 (SEQ ID NOs: (a) 470, 471 and/or 472, or (b) 604, 605 and/or 606), cdrCAN46G24 (SEQ ID NOs: (a) 486, 487 and/or 488, or (b) 620, 621 and/or 622), huCAN46G24 (SEQ ID NOs: (a) 502, 503 and/or 504, or (b) 636, 637 and/or 638) or rehuCAN46G24 (SEQ ID NOs: (a) 518, 519 and/or 520, or (b) 652, 653 and/or 654); or, (ii) the cdrs of the light chain variable region as set forth in cdrCAN46G4 (SEQ ID NOs: 660, 661 and/or 662), huCAN46G4 (SEQ ID NOs: 676, 677 and/or 678), rehuCAN46G4 (SEQ ID NOs: 692, 693 and/or 694), cdrCAN46G13a (SEQ ID NOs: (a) 382 383 and/or 384, or (b) 526, 527 and/or 528), huCAN46G13a (SEQ ID NOs: (a) 398, 399 and/or 400, or (b) 542, 543 and/or 544), rehuCAN46G13a (SEQ ID NOs: (a) 414, 415 and/or 416, or (b) 558, 559 and/or 560), cdrCAN46G19 (SEQ ID NOs: (a) 430, 431 and/or 432, or (b) 715, 716 and/or 717), huCAN46G19 (SEQ ID NOs: (a) 446, 447 and/or 448, or (b) 580, 581 and 582), rehuCAN46G19 (SEQ ID NOs: (a) 462, 463 and/or 464, or (b) 596, 597 and/or 598), cdrCAN46G24 (SEQ ID NOs: (a) 478, 479 and/or 480, or (b) 612, 613 and/or 614), huCAN46G24 (SEQ ID NOs: (a) 494, 495 and/or 496 or (b) 628, 629 and/or 630) or rehuCAN46G24 (SEQ ID NOs: (a) 510, 511 and 512, or (b) 644, 645 and/or 646).

In certain embodiments, the antibody or antigen-binding portions comprise both heavy chain variable regions (i) and light chain variable regions (ii). Specifically, The antibodies or antigen-binding portion can comprise one, two, three or more complementarity determining regions (CDRs) encoded by nucleic acid sequences that are homologous to one, two, three or more CDRs encoded by the nucleic acid sequence of: (i) the cdrs of the heavy chain variable region as set forth in, cdrCAN46G4 (SEQ ID NOs: 668, 669 and/or 670), huCAN46G4 (SEQ ID NOs: 684, 685 and/or 686), rehuCAN46G4 (SEQ ID NOs: 700, 701 and/or 702), cdrCAN46G13a (SEQ ID NOs: (a) 390, 391 and/or 392, or (b) 534, 535 and/or 536), huCAN46G13a (SEQ ID NOs: (a) 406, 407 and/or 408, or (b) 550, 551 and/or 552), rehuCAN46G13a (SEQ ID NOs: (a) 422, 423 and/or 424, or (b) 566, 567 and/or 568), cdrCAN46G19 (SEQ ID NOs: (a) 438, 439 and 440, or (b) 572, 573 and/or 574), huCAN46G19 (SEQ ID NOs: (a) 454, 455 and/or 456, or (b) 588, 589 and/or 590), rehuCAN46G19 (SEQ ID NOs: (a) 470, 471 and/or 472, or (b) 604, 605 and/or 606), cdrCAN46G24 (SEQ ID NOs: (a) 486, 487 and/or 488, or (b) 620, 621 and/or 622), huCAN46G24 (SEQ ID NOs: (a) 502, 503 and/or 504, or (b) 636, 637 and/or 638) or rehuCAN46G24 (SEQ ID NOs: (a) 518, 519 and/or 520, or (b) 652, 653 and/or 654); and, (ii) the cdrs of the light chain variable region as set forth in cdrCAN46G4 (SEQ ID NOs: 660, 661 and/or 662), huCAN46G4 (SEQ ID NOs: 676, 677 and/or 678), rehuCAN46G4 (SEQ ID NOs: 692, 693 and/or 694), cdrCAN46G13a (SEQ ID NOs: (a) 382 383 and/or 384, or (b) 526, 527 and/or 528), huCAN46G13a (SEQ ID NOs: (a) 398, 399 and/or 400, or (b) 542, 543 and/or 544), rehuCAN46G13a (SEQ ID NOs: (a) 414, 415 and/or 416, or (b) 558, 559 and/or 560), cdrCAN46G19 (SEQ ID NOs: (a) 430, 431 and/or 432, or (b) 715, 716 and/or 717), huCAN46G19 (SEQ ID NOs: (a) 446, 447 and/or 448, or (b) 580, 581 and 582), rehuCAN46G19 (SEQ ID NOs: (a) 462, 463 and/or 464, or (b) 596, 597 and/or 598), cdrCAN46G24 (SEQ ID NOs: (a) 478, 479 and/or 480, or (b) 612, 613 and/or 614), huCAN46G24 (SEQ ID NOs: (a) 494, 495 and/or 496 or (b) 628, 629 and/or 630) or rehuCAN46G24 (SEQ ID NOs: (a) 510, 511 and 512, or (b) 644, 645 and/or 646). The antibodies or antigen-binding portion can comprise any combination, one, two, three or more, of the CDRs encoded by nucleic acid sequences for the heavy chain variable and light chain variable set forth in the preceeding paragraph.

The antibodies or antigen-binding portion can comprise: (i) a heavy chain variable region comprising three CDRs that are encoded by nucleic acid sequences homologous to nucleic acid sequences as set forth in cdrCAN46G4 (SEQ ID NOs: 668, 669 and 670), huCAN46G4 (SEQ ID NOs: 684, 685 and 686), rehuCAN46G4 (SEQ ID NOs: 700, 701 and 702), cdrCAN46G13a (SEQ ID NOs: (a) 390, 391 and 392; or (b) 534, 535 and 536), huCAN46G13a (SEQ ID NOs: (a) 406, 407 and 408, or (b) 550, 551 and 552), rehuCAN46G13a (SEQ ID NOs: (a) 422, 423 and 424, or (b) 566, 567 and 568), cdrCAN46G19 (SEQ ID NOs: (a) 438, 439 and 440, or (b) 572, 573 and 574), huCAN46G19 (SEQ ID NOs: (a) 454, 455 and 456, or (b) 588, 589 and 590), rehuCAN46G19 (SEQ ID NOs: (a) 470, 471 and 472, or (b) 604, 605 and 606), cdrCAN46G24 (SEQ ID NOs: (a) 486, 487 and 488, or (b) 620, 621 and 622), huCAN46G24 (SEQ ID NOs: (a) 502, 503 and 504, or (b) 636, 637 and 638) or rehuCAN46G24 (SEQ ID NOs: (a) 518, 519 and 520, or (b) 652, 653 and 654); or, (ii) a light chain variable region comprising three CDRs that are encoded by nucleic acid sequences homologous to nucleic acid sequences as set forth in cdrCAN46G4 (SEQ ID NOs: 660, 661 and 662), huCAN46G4 (SEQ ID NOs: 676, 677 and 678), rehuCAN46G4 (SEQ ID NOs: 692, 693 and 694), cdrCAN46G13a (SEQ ID NOs: (a) 382 383 and 384, or (b) 526, 527 and 528), huCAN46G13a (SEQ ID NOs: (a) 398, 399 and 400, or (b) 542, 543 and 544), rehuCAN46G13a (SEQ ID NOs: (a) 414, 415 and 416, or (b) 558, 559 and 560), cdrCAN46G19 (SEQ ID NOs: (a) 430, 431 and 432, or (b) 715, 716 and 717), huCAN46G19 (SEQ ID NOs: (a) 446, 447 and 448, or (b) 580, 581 and 582), rehuCAN46G19 (SEQ ID NOs: (a) 462, 463 and 464, or (b) 596, 597 and 598), cdrCAN46G24 (SEQ ID NOs: (a) 478, 479 and 480, or (b) 612, 613 and 614), huCAN46G24 (SEQ ID NOs: (a) 494, 495 and 496, or (b) 628, 629 and 630) or rehuCAN46G24 (SEQ ID NOs: (a) 510, 511 and 512, or (b) 644, 645 and 646).

In certain embodiments, the antibody or antigen-binding portions comprise both heavy chain variable regions (i) and light chain variable regions (ii). Specifically, the heavy and light chain variable regions comprise three CDRs that are encoded by nucleic acid sequences homologous to nucleic acid sequences as set forth in: (i) a heavy chain variable region comprising three CDRs that are encoded by nucleic acid sequences homologous to nucleic acid sequences as set forth in cdrCAN46G4 (SEQ ID NOs: 668, 669 and 670), huCAN46G4 (SEQ ID NOs: 684, 685 and 686), rehuCAN46G4 (SEQ ID NOs: 700, 701 and 702), cdrCAN46G13a (SEQ ID NOs: (a) 390, 391 and 392; or (b) 534, 535 and 536), huCAN46G13a (SEQ ID NOs: (a) 406, 407 and 408, or (b) 550, 551 and 552), rehuCAN46G13a (SEQ ID NOs: (a) 422, 423 and 424, or (b) 566, 567 and 568), cdrCAN46G19 (SEQ ID NOs: (a) 438, 439 and 440, or (b) 572, 573 and 574), huCAN46G19 (SEQ ID NOs: (a) 454, 455 and 456, or (b) 588, 589 and 590), rehuCAN46G19 (SEQ ID NOs: (a) 470, 471 and 472, or (b) 604, 605 and 606), cdrCAN46G24 (SEQ ID NOs: (a) 486, 487 and 488, or (b) 620, 621 and 622), huCAN46G24 (SEQ ID NOs: (a) 502, 503 and 504, or (b) 636, 637 and 638) or rehuCAN46G24 (SEQ ID NOs: (a) 518, 519 and 520, or (b) 652, 653 and 654); or, (ii) a light chain variable region comprising three CDRs that are encoded by nucleic acid sequences homologous to nucleic acid sequences as set forth in cdrCAN46G4 (SEQ ID NOs: 660, 661 and 662), huCAN46G4 (SEQ ID NOs: 676, 677 and 678), rehuCAN46G4 (SEQ ID NOs: 692, 693 and 694), cdrCAN46G13a (SEQ ID NOs: (a) 382 383 and 384, or (b) 526, 527 and 528), huCAN46G13a (SEQ ID NOs: (a) 398, 399 and 400, or (b) 542, 543 and 544), rehuCAN46G13a (SEQ ID NOs: (a) 414, 415 and 416, or (b) 558, 559 and 560), cdrCAN46G19 (SEQ ID NOs: (a) 430, 431 and 432, or (b) 715, 716 and 717), huCAN46G19 (SEQ ID NOs: (a) 446, 447 and 448, or (b) 580, 581 and 582), rehuCAN46G19 (SEQ ID NOs: (a) 462, 463 and 464, or (b) 596, 597 and 598), cdrCAN46G24 (SEQ ID NOs: (a) 478, 479 and 480, or (b) 612, 613 and 614), huCAN46G24 (SEQ ID NOs: (a) 494, 495 and 496, or (b) 628, 629 and 630) or rehuCAN46G24 (SEQ ID NOs: (a) 510, 511 and 512, or (b) 644, 645 and 646). The antibodies or antigen-binding portions can comprise any combination of the CDRS encoded for by the nucleic acid sequences set forth for the heavy chain variable region and light chain variable region listed in the preceeding paragraph.

In one embodiment, the antibody or antigen-binding portion contains a light chain variable region and heavy chain variable region encoded by nucleic acid sequences homologous to nucleic acid sequences as set forth in cdrCAN46G4 (SEQ ID NOs: 659 and 667, light chain variable and heavy chain variable region, respectively), huCAN46G4 (SEQ ID NOs: 675 and 683, light chain variable and heavy chain variable region, respectively), rehuCAN46G4 (691 and 699, light chain variable and heavy chain variable region, respectively), cdrCAN46G13a (SEQ ID NOs: (a) 381 and 389, or (b) 525 and 533, respectively), huCAN46G13a (SEQ ID NOs: (a) 397 and 405, or (b) 541 and 549, light chain variable and heavy chain variable region, respectively), rehuCAN46G13a (SEQ ID NOs: (a) 413 and 421, or (b) 557 and 565, light chain variable and heavy chain variable region, respectively), cdrCAN46G19 (SEQ ID NOs: (a) 429 and 437, or (b) 714 and 571 light chain variable and heavy chain variable region, respectively), huCAN46G19 (SEQ ID NOs: (a) 445 and 453, or (b) 579 and 587, light chain variable and heavy chain variable region, respectively), rehuCAN46G19 (SEQ ID NOs: (a) 461 and 469, or (b) 595 and 603, light chain variable and heavy chain variable region, respectively), cdrCAN46G24 (SEQ ID NOs: (a) 477 and 485, or (b) 611 and 619, light chain variable and heavy chain variable region, respectively), huCAN46G24 (SEQ ID NOs: (a) 493 and 501, or (b) 627 and 635, light chain variable and heavy chain variable region, respectively) or rehuCAN46G24 (SEQ ID NOs: (a) 509 and 517, or (b) 643 and 651, light chain variable and heavy chain variable region, respectively). The antibodies or antigen-binding portions can comprise any combination of the nucleic sequences set forth for the heavy chain variable region (1) and light chain variable region (2) listed in the preceeding paragraph.

In another embodiment, the antibody or antigen-binding portion contains a light chain variable region encoded by nucleic acid sequences homologous to nucleic acid sequences as set forth in CAN46G4 (SEQ ID NOs: 659, 675, or 691), CAN46G13a (SEQ ID NOs: 381, 397, 413, 525, 541, 557, or 707), CAN46G19 (SEQ ID NOs: 429, 445, 461, 714, 579, or 595), or CAN46G24 (SEQ ID NOs: 477, 493, 509, 611, 627, or 643).

The antibody or antigen-binding portion comprises a heavy chain variable region encoded by nucleic acid sequences homologous to nucleic acid sequences as set forth in CAN46G4 (SEQ ID NOs: 667, 683, or 699), CAN46G13a (SEQ ID NOs: 389, 405, 421, 533, 549, 565, or 709), CAN46G19 (SEQ ID NOs: 437, 453, 469, 571, 587, or 603), or CAN46G24 (SEQ ID NOs: 485, 501, 517, 619, 635, or 651).

Humanized Antibodies

The humanized antibody of the present invention is an antibody from a non-human species where the amino acid sequence in the non-antigen-binding regions (and/or the antigen-binding regions) has been altered so that the antibody more closely resembles a human antibody, and still retains comparable specificity and affinity.

Humanized antibodies can be generated by replacing sequences of the variable region that are not directly involved in antigen-binding with equivalent sequences from human variable regions. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against toxin B. The recombinant DNA encoding the humanized antibody, or fragment, can then be cloned into an appropriate expression vector.

An antibody light or heavy chain variable region consists of a framework region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs). In one embodiment, humanized antibodies are antibody molecules from non-human species having one, two or all CDRs from the non-human species and a framework region from a human immunoglobulin molecule.

The humanized antibodies of the present invention can be produced by methods known in the art. For example, once non-human (e.g., murine) antibodies are obtained, variable regions can be sequenced, and the location of the CDRs and framework residues determined. Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. Chothia, C. et al. (1987) J. Mol. Biol., 196:901-917. The light and heavy chain variable regions can, optionally, be ligated to corresponding constant regions. CDR-grafted antibody molecules can be produced by CDR-grafting or CDR substitution. One, two, or all CDRs of an immunoglobulin chain can be replaced. For example, all of the CDRs of a particular antibody may be from at least a portion of a non-human animal (e.g., mouse such as CDRs shown in Table 1) or only some of the CDRs may be replaced. It is only necessary to keep the CDRs required for binding of the antibody to a predetermined antigen (e.g., toxin B of C. difficile). Morrison, S. L., 1985, Science, 229:1202-1207. Oi et al., 1986, BioTechniques, 4:214. U.S. Pat. Nos. 5,585,089; 5,225,539; 5,693,761 and 5,693,762. EP 519596. Jones et al., 1986, Nature, 321:552-525. Verhoeyan et al., 1988, Science, 239:1534. Beidler et al., 1988, J. Immunol., 141:4053-4060.

Also encompassed by the present invention are antibodies or antigen-binding portion containing one, two, or all CDRs as disclosed herein, with the other regions replaced by sequences from at least one different species including, but not limited to, human, rabbits, sheep, dogs, cats, cows, horses, goats, pigs, monkeys, apes, gorillas, chimpanzees, ducks, geese, chickens, amphibians, reptiles and other animals.

Chimeric Antibodies

A chimeric antibody is a molecule in which different portions are derived from different animal species. For example, an antibody may contain a variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies can be produced by recombinant DNA techniques. Morrison, et al., Proc Natl Acad Sci, 81:6851-6855 (1984). For example, a gene encoding a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted. Chimeric antibodies can also be created by recombinant DNA techniques where DNA encoding murine V regions can be ligated to DNA encoding the human constant regions. Better et al., Science, 1988, 240:1041-1043. Liu et al. PNAS, 1987 84:3439-3443. Liu et al., J. Immunol., 1987, 139:3521-3526. Sun et al. PNAS, 1987, 84:214-218. Nishimura et al., Canc. Res., 1987, 47:999-1005. Wood et al. Nature, 1985, 314:446-449. Shaw et al., J. Natl. Cancer Inst., 1988, 80:1553-1559. International Patent Publication Nos. WO1987002671 and WO 86/01533. European Patent Application Nos. 184, 187; 171,496; 125,023; and 173,494. U.S. Pat. No. 4,816,567.

Types of Antibodies

The antibodies can be full-length or can include a fragment (or fragments) of the antibody having an antigen-binding portion, including, but not limited to, Fab, F(ab′)2, Fab′, F(ab)′, Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragment (e.g., Ward et al., Nature, 341:544-546 (1989)), an isolated CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker, are also encompassed by the present invention. Bird et al. Science, 1988, 242:423-426. Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883.

The antibodies or antigen-binding portion of the present invention may be monospecific, bi-specific or multispecific. Multispecific or bi-specific antibodies or fragments thereof may be specific for different epitopes of one target polypeptide (e.g., toxin B) or may contain antigen-binding domains specific for more than one target polypeptide (e.g., antigen-binding domains specific for toxin A and toxin B; antigen-binding domains specific for toxin B and other antigen of C. difficile; or antigen-binding domains specific for toxin B and other kind of bacterium or virus). In one embodiment, a multispecific antibody or antigen-binding portion thereof comprises at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Tutt et al., 1991, J. Immunol. 147:60-69. Kufer et al., 2004, Trends Biotechnol. 22:238-244. The present antibodies can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second binding specificity. For example, the present invention includes bi-specific antibodies wherein one arm of an immunoglobulin is specific for toxin B, and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety such as a trypsin inhibitor.

All antibody isotypes are encompassed by the present invention, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE. The antibodies or antigen-binding portion may be mammalian (e.g., mouse, human) antibodies or antigen-binding portion. The light chains of the antibody may be of kappa or lambda type. The antibodies or antigen-binding portion may also be based on camelid (Bactrian camels, dromedaries and llamas) antibodies devoid of light chains also referred to as Nanobody®.

Variations of the Antibodies

The antibodies or antigen-binding portion are peptides. The peptides may also include variants, analogs, orthologs, homologs and derivatives of peptides, that exhibit a biological activity, e.g., binding of an antigen. The peptides may contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), peptides with substituted linkages, as well as other modifications known in the art.

Also within the scope of the invention are antibodies or antigen-binding portion in which specific amino acids have been substituted, deleted or added. These alternations do not have a substantial effect on the peptide's biological properties such as binding activity. For example, antibodies may have amino acid substitutions in the framework region, such as to improve binding to the antigen, modify solubility and/or influence pharmacokinetics/pharmacodynamics. In another example, a selected, small number of acceptor framework residues can be replaced by the corresponding donor amino acids. The donor framework can be a mature or germline human antibody framework sequence or a consensus sequence. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science, 247: 1306-1310 (1990). Cunningham et al., Science, 244: 1081-1085 (1989). Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994). T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989). Pearson, Methods Mol. Biol. 243:307-31 (1994). Gonnet et al., Science 256:1443-45 (1992).

The antibody, or antigen-binding portion can be derivatized or linked to another functional molecule. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent interaction, etc.) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or a polyhistidine tag), amino acid linkers, signal sequences, immunogenic carriers, or ligands useful in protein purification, such as glutathione-S-transferase, histidine tag, and staphylococcal protein A. One type of derivatized protein is produced by crosslinking two or more proteins (of the same type or of different types). Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill. Useful detectable agents with which a protein can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting, exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, and, phycoerythrin. A protein or antibody can also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase and the like. A protein can also be derivatized with a prosthetic group (e.g., streptavidin/biotin and avidin/biotin).

The present peptides may be the functionally active variant of antibodies of antigen-binding portion disclosed herein, e.g., with less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 1% amino acid residues substituted or deleted but retain essentially the same immunological properties including, but not limited to, binding to toxin B.

The antibody, or antigen-binding portion thereof, can be codon optimized. For example, codons within the cloned gene that are generally not used by the host cell translation system are changed by in vitro mutagenesis to the preferred codons of the host cell system without changing the amino acid sequence of the synthesized antibody.

Nucleic Acids Encoding Antibody Variable Regions

The invention also encompasses a nucleic acid encoding the present antibody or antigen-binding portion thereof that specifically binds to toxin B of C. difficile. The nucleic acid may be expressed in a cell to produce the present antibody or antigen-binding portion thereof. The isolated nucleic acid of the present invention comprises a sequence encoding a peptide homologous to SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 85 or 93.

The invention also encompasses expression vectors including: (i) a nucleic acid encoding a peptide homologous to amino acid sequences SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 85 or 93; (ii) a nucleic acid encoding a peptide homologous to amino acid SEQ ID NOs: 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229, 237, 708 and 710; or (iii) a nucleic acid encoding a peptide homologous to nucleic acid sequences SEQ ID NOs: 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 477, 485, 493, 501, 509, 517, 525, 533, 541, 549, 565, 557, 714, 571, 579, 587, 595, 603, 611, 619, 627, 635, 643 and 651. The nucleic acid may be expressed in a cell to produce the present antibody or antigen-binding portion thereof.

Nucleic acid molecules encoding a functionally active variant of the present antibody or antigen-binding portion thereof are also encompassed by the present invention. These nucleic acid molecules may hybridize with a nucleic acid encoding any of the present antibody or antigen-binding portion thereof under medium stringency, high stringency, or very high stringency conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which is incorporated herein by reference. Specific hybridization conditions referred to herein are as follows: 1) medium stringency hybridization conditions: 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 2) high stringency hybridization conditions: 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and 3) very high stringency hybridization conditions: 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.

A nucleic acid encoding the present antibody or antigen-binding portion may be introduced into an expression vector that can be expressed in a suitable expression system, followed by isolation or purification of the expressed antibody or antigen-binding portion thereof. Optionally, a nucleic acid encoding the present antibody or antigen-binding portion thereof can be translated in a cell-free translation system. U.S. Pat. No. 4,816,567. Queen et al., Proc Natl Acad Sci USA, 86:10029-10033 (1989).

Anti-toxin antibodies or portions can be produced by host cells transformed with DNA encoding light and heavy chains (or portions thereof) of a desired antibody. Antibodies can be isolated and purified from these culture supernatants and/or cells using standard techniques. For example, a host cell may be transformed with DNA encoding the light chain, the heavy chain, or both, of an antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding, e.g., the constant region.

The present nucleic acids can be expressed in various suitable cells, including prokaryotic and eukaryotic cells, e.g., bacterial cells, (e.g., E. coli), yeast cells, plant cells, insect cells, and mammalian cells. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC). Non-limiting examples of the cells include all cell lines of mammalian origin or mammalian-like characteristics, including but not limited to, parental cells, derivatives and/or engineered variants of monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO, e.g. CHOSKV1), NS0, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2/0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells. The engineered variants include, e.g., glycan profile modified and/or site-specific integration site derivatives.

The present invention also provides for cells comprising the nucleic acids described herein. The cells may be a hybridoma or transfectant. The types of the cells are discussed above.

The present antibody or antigen-binding portion can be expressed in various cells. The types of the cells are discussed above.

The present antibody or antigen-binding portion thereof can be expressed in cell-free translation systems, viral infection constructs or transgenic animals.

Alternatively, the present antibody or antigen-binding portion can be synthesized by solid phase procedures well known in the art. Solid Phase Peptide Synthesis: A Practical Approach by E. Atherton and R. C. Sheppard, published by IRL at Oxford University Press (1989). Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed. M. W. Pennington and B. M. Dunn), chapter 7. Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984). G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 1 and Vol. 2, Academic Press, New York, (1980), pp. 3-254. M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984).

C. difficile Toxins

The present invention provides for methods for making an antibody or antigen-binding portion thereof that specifically binds to toxin B of C. difficile. For example, a non-human animal is immunized with a composition that includes an inactivated toxin B, toxoid B, fragment of ToxinB, modified fragment of toxinB (synthetic variant), and then a specific antibody is isolated from the animal. The method can further include evaluating binding of the antibody to toxin B.

Any of a variety of Clostridium difficile toxin proteins, particularly toxin B, may be used in the practice of the present invention. In one embodiment toxin B is isolated from strain VPI10463. Toxin A and toxin B of C. difficile are high molecular mass proteins (280 to 310 kDa) that possess multiple functional domains, also referred to as fragments or domains. The N-terminal domains of both toxins contain glucosyltransferase activity that modifies Rho-like GTPases. This modification leads to cytoskeletal dysregulation in the toxified cells and disruption of colonic epithelial tight junctions. The central domain is predicted to be involved in membrane transport given the presence of hydrophobic regions and caveolin binding sites. The C-terminal third of the toxins contains repeating subunits believed to interact with carbohydrate receptors expressed on the target cell surface. The interaction of toxin A with carbohydrates also induces the hemagglutination of rabbit erythrocytes and provides a model for the study of toxin A receptor binding. Both toxins are cytotoxic, with toxin B being 1000 times more potent than toxin A when tested in in vitro cytotoxicity assays, and both are lethal when injected intravenously or intraperitoneally (i.p.) into a mouse. Toxin A is also a potent enterotoxin, as demonstrated by the induction of fluid accumulation in the mouse ligated intestinal loop diarrhea model. See, e.g., Babcock, G. J. et al., Infection and Immunity, 74: 6339-6347 (2006) and references contained therein for background.

Table 2 provides amino acid sequences of Clostridium difficile toxin B. Variants and fragments of the sequences provided below can also be used as an antigen to generate antibodies.

TABLE 2 Accession Number SEQ And ID Protein NO Name Amino acid Sequence 83 NC_009089, MSLVNRKQLEKMANVRFRTQEDEYVAILDALEEYHNMSEN Toxin B TVVEKYLKLKDINSLTDIYIDTYKKSGRNKALKKFKEYLVTE (tcdB) VLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKDVNS DYNVNVFYDSNAFLINTLKKTVVESAINDTLESFRENLNDPR FDYNKFFRKRMEIIYDKQKNFINYYKAQREENPELIIDDIVKT YLSNEYSKEIDELNTYIEESLNKITQNSGNDVRNFEEFKNGES FNLYEQELVERWNLAAASDILRISALKEIGGMYLDVDMLPGI QPDLFESIEKPSSVTVDFWEMTKLEAIMKYKEYIPEYTSEHFD MLDEEVQSSFESVLASKSDKSEIFSSLGDMEASPLEVKIAFNS KGIINQGLISVKDSYCSNLIVKQIENRYKILNNSLNPAISEDND FNTTTNTFIDSIMAEANADNGRFMMELGKYLRVGFFPDVKT TINLSGPEAYAAAYQDLLMFKEGSMNIHLIEADLRNFEISKTN ISQSTEQEMASLWSFDDARAKAQFEEYKRNYFEGSLGEDDN LDFSQNIVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYE AACNLFAKTPYDSVLFQKNIEDSEIAYYYNPGDGEIQEIDKY KIPSIISDRPKIKLTFIGHGKDEFNTDIFAGFDVDSLSTEIEAAID LAKEDISPKSIEINLLGCNMFSYSINVEETYPGKLLLKVKDKIS ELMPSISQDSIIVSANQYEVRINSEGRRELLDHSGEWINKEESII KDISSKEYISFNPKENKITVKSKNLPELSTLLQEIRNNSNSSDIE LEEKVMLTECEINVISNIDTQIVEERIEEAKNLTSDSINYIKDEF KLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRFINKE TGESIFVETEKTIFSEYANHITEEISKIKGTIFDTVNGKLVKKV NLDTTHEVNTLNAAFFIQSLIEYNSSKESLSNLSVAMKVQVY AQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPIIATI IDGVSLGAAIKELSETSDPLLRQEIEAKIGIMAVNLTTATTAIIT SSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYF KHVSLVETEGVFTLLDDKIMMPQDDLVISEIDFNNNSIVLGK CEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHLSIYDVLEVQ KEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKL LDRIRDNYEGEFYWRYFAFIADALITTLKPRYEDTNIRINLDS NTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIEL SESDVWIIDVDNVVRDVTIESDKIKKGDLIEGILSTLSIEENKII LNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLL ISGELKILMLNSNHIQQKIDYIGFNSELQKNIPYSFVDSEGKEN GFINGSTKEGLFVSELPDVVLISKVYMDDSKPSFGYYSNNLK DVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHL DESGVAEILKFMNRKGNTNTSDSLMSFLESMNIKSIFVNFLQS NIKFILDANFIISGTTSIGQFEFICDENDNIQPYFIKFNTLETNYT LYVGNRQNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCV NKVVISPNIYTDEINITPVYETNNTYPEVIVLDANYINEKINVN INDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDK TLANKLSFNFSDKQDVPVSEIILSFTPSYYEDGLIGYDLGLVSL YNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNLITGFVTVG DDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTE DGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRG AVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGV MQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGE MQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKI YYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDG QYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYF YIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGL VRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKAC KGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFG YINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFE GESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPD TAQLVISE

Table 3 provides nucleic acid sequences encoding the proteins of Table 2.

TABLE 3 Accession  SEQ Number ID And Gene NO Name Nucleotide Sequence 84 NC_009089, atgagtttagttaatagaaaacagttagaaaaaatggcaaatgtaagatttcgtactcaagaagatg Toxin B aatatgttgcaatattggatgctttagaagaatatcataatatgtcagagaatactgtagtcgaaaaat tcdB atttaaaattaaaagatataaatagtttaacagatatttatatagatacatataaaaaatctggtagaaat aaagccttaaaaaaatttaaggaatatctagttacagaagtattagagctaaagaataataatttaact ccagttgagaaaaatttacattttgtttggattggaggtcaaataaatgacactgctattaattatataa atcaatggaaagatgtaaatagtgattataatgttaatgttttttatgatagtaatgcatttttgataaaca cattgaaaaaaactgtagtagaatcagcaataaatgatacacttgaatcatttagagaaaacttaaat gaccctagatttgactataataaattcttcagaaaacgtatggaaataatttatgataaacagaaaaat ttcataaactactataaagctcaaagagaagaaaatcctgaacttataattgatgatattgtaaagac atatctttcaaatgagtattcaaaggagatagatgaacttaatacctatattgaagaatccttaaataaa attacacagaatagtggaaatgatgttagaaactttgaagaatttaaaaatggagagtcattcaactt atatgaacaagagttggtagaaaggtggaatttagctgctgcttctgacatattaagaatatctgcatt aaaagaaattggtggtatgtatttagatgttgatatgttaccaggaatacaaccagacttatttgagtct atagagaaacctagttcagtaacagtggatttttgggaaatgacaaagttagaagctataatgaaat acaaagaatatataccagaatatacctcagaacattttgacatgttagacgaagaagttcaaagtag ttttgaatctgttctagcttctaagtcagataaatcagaaatattctcatcacttggtgatatggaggcat caccactagaagttaaaattgcatttaatagtaagggtattataaatcaagggctaatttctgtgaaag actcatattgtagcaatttaatagtaaaacaaatcgagaatagatataaaatattgaataatagtttaaa tccagctattagcgaggataatgattttaatactacaacgaatacctttattgatagtataatggctgaa gctaatgcagataatggtagatttatgatggaactaggaaagtatttaagagttggtttcttcccagat gttaaaactactattaacttaagtggccctgaagcatatgcggcagcttatcaagatttattaatgttta aagaaggcagtatgaatatccatttgatagaagctgatttaagaaactttgaaatctctaaaactaa tatttctcaatcaactgaacaagaaatggctagcttatggtcatttgacgatgcaagagctaaagctc aatttgaagaatataaaaggaattattttgaaggttctcttggtgaagatgataatcttgatttttctcaa aatatagtagttgacaaggagtatcttttagaaaaaatatcttcattagcaagaagttcagagagagg atatatacactatattgttcagttacaaggagataaaattagttatgaagcagcatgtaacttatttgca aagactccttatgatagtgtactgtttcagaaaaatatagaagattcagaaattgcatattattataatc ctggagatggtgaaatacaagaaatagacaagtataaaattccaagtataatttctgatagacctaa gattaaattaacatttattggtcatggtaaagatgaatttaatactgatatatttgcaggttttgatgtaga ttcattatccacagaaatagaagcagcaatagatttagctaaagaggatatttctcctaagtcaatag aaataaatttattaggatgtaatatgtttagctactctatcaacgtagaggagacttatcctggaaaatt attacttaaagttaaagataaaatatcagaattaatgccatctataagtcaagactctattatagtaagt gcaaatcaatatgaagttagaataaatagtgaaggaagaagagaattattggatcattctggtgaat ggataaataaagaagaaagtattataaaggatatttcatcaaaagaatatatatcatttaatcctaaag aaaataaaattacagtaaaatctaaaaatttacctgagctatctacattattacaagaaattagaaata attctaattcaagtgatattgaactagaagaaaaagtaatgttaacagaatgtgagataaatgttatttc aaatatagatacgcaaattgttgaggaaaggattgaagaagctaagaatttaacttctgactctatta attatataaaagatgaatttaaactaatagaatctatttctgatgcactatgtgacttaaaacaacagaa tgaattagaagattctcattttatatcttttgaggacatatcagagactgatgagggatttagtataaga tttattaataaagaaactggagaatctatatttgtagaaactgaaaaaacaatattctctgaatatgcta atcatataactgaagagatttctaagataaaaggtactatatttgatactgtaaatggtaagttagtaaa aaaagtaaatttagatactacacacgaagtaaatactttaaatgctgcattttttatacaatcattaatag aatataatagttctaaagaatctcttagtaatttaagtgtagcaatgaaagtccaagtttacgctcaatt atttagtactggtttaaatactattacagatgcagccaaagttgttgaattagtatcaactgcattagat gaaactatagacttacttectacattatctgaaggattacctataattgcaactattatagatggtgtaa gtttaggtgcagcaatcaaagagctaagtgaaacgagtgacccattattaagacaagaaatagaa gctaagataggtataatggcagtaaatttaacaacagctacaactgcaatcattacttcatctttggg gatagctagtggatttagtatacttttagttcctttagcaggaatttcagcaggtataccaagcttagta aacaatgaacttgtacttcgagataaggcaacaaaggttgtagattattttaaacatgtttcattagttg aaactgaaggagtatttactttattagatgataaaataatgatgccacaagatgatttagtgatatcag aaatagattttaataataattcaatagttttaggtaaatgtgaaatctggagaatggaaggtggttcag gtcatactgtaactgatgatatagatcacttcttttcagcaccatcaataacatatagagagccacact tatctatatatgacgtattggaagtacaaaaagaagaacttgatttgtcaaaagatttaatggtattacc taatgctccaaatagagtatttgcttgggaaacaggatggacaccaggtttaagaagcttagaaaat gatggcacaaaactgttagaccgtataagagataactatgaaggtgagttttattggagatattttgct tttatagctgatgctttaataacaacattaaaaccaagatatgaagatactaatataagaataaatttag atagtaatactagaagttttatagttccaataataactacagaatatataagagaaaaattatcatattct ttctatggttcaggaggaacttatgcattgtctctttctcaatataatatgggtataaatatagaattaag tgaaagtgatgtttggattatagatgttgataatgttgtgagagatgtaactatagaatctgataaaatt aaaaaaggtgatttaatagaaggtattttatctacactaagtattgaagagaataaaattatcttaaata gccatgagattaatttttctggtgaggtaaatggaagtaatggatttgtttctttaacattttcaattttag aaggaataaatgcaattatagaagttgatttattatctaaatcatataaattacttatttctggcgaatta aaaatattgatgttaaattcaaatcatattcaacagaaaatagattatataggattcaatagcgaattac agaaaaatataccatatagctttgtagatagtgaaggaaaagagaatggttttattaatggttcaaca aaagaaggtttatttgtatctgaattacctgatgtagttcttataagtaaggtttatatggatgatagtaa gccttcatttggatattatagtaataatttgaaagatgtcaaagttataactaaagataatgttaatatatt aacaggttattatcttaaggatgatataaaaatctctctttctttgactctacaagatgaaaaaactata aagttaaatagtgtgcatttagatgaaagtggagtagctgagattttgaagttcatgaatagaaaagg taatacaaatacttcagattctttaatgagctttttagaaagtatgaatataaaaagtattttcgttaatttc ttacaatctaatattaagtttatattagatgctaattttataataagtggtactacttctattggccaatttg agtttatttgtgatgaaaatgataatatacaaccatatttcattaagtttaatacactagaaactaattata ctttatatgtaggaaatagacaaaatatgatagtggaaccaaattatgatttagatgattctggagata tatcttcaactgttatcaatttctctcaaaagtatctttatggaatagacagttgtgttaataaagttgtaa tttcaccaaatatttatacagatgaaataaatataacgcctgtatatgaaacaaataatacttatccaga agttattgtattagatgcaaattatataaatgaaaaaataaatgttaatatcaatgatctatctatacgat atgtatggagtaatgatggtaatgattttattcttatgtcaactagtgaagaaaataaggtgtcacaag ttaaaataagattcgttaatgtttttaaagataagactttggcaaataagctatcttttaactttagtgata aacaagatgtacctgtaagtgaaataatcttatcatttacaccttcatattatgaggatggattgattgg ctatgatttgggtctagtttctttatataatgagaaattttatattaataactttggaatgatggtatctgga ttaatatatattaatgattcattatattattttaaaccaccagtaaataatttgataactggatttgtgactg taggcgatgataaatactactttaatccaattaatggtggagctgcttcaattggagagacaataatt gatgacaaaaattattatttcaaccaaagtggagtgttacaaacaggtgtatttagtacagaagatgg atttaaatattttgccccagctaatacacttgatgaaaacctagaaggagaagcaattgattttactgg aaaattaattattgacgaaaatatttattattttgatgataattatagaggagctgtagaatggaaagaa ttagatggtgaaatgcactattttagcccagaaacaggtaaagcttttaaaggtctaaatcaaatagg tgattataaatactatttcaattctgatggagttatgcaaaaaggatttgttagtataaatgataataaac actattttgatgattctggtgttatgaaagtaggttacactgaaatagatggcaagcatttctactttgct gaaaacggagaaatgcaaataggagtatttaatacagaagatggatttaaatattttgctcatcataa tgaagatttaggaaatgaagaaggtgaagaaatctcatattctggtatattaaatttcaataataaaatt tactattttgatgattcatttacagctgtagttggatggaaagatttagaggatggttcaaagtattatttt gatgaagatacagcagaagcatatataggtttgtcattaataaatgatggtcaatattattttaatgatg atggaattatgcaagttggatttgtcactataaatgataaagtcttctacttctctgactctggaattata gaatctggagtacaaaacatagatgacaattatttctatatagatgataatggtatagttcaaattggt gtatttgatacttcagatggatataaatattttgcacctgctaatactgtaaatgataatatttacggaca agcagttgaatatagtggtttagttagagttggtgaagatgtatattattttggagaaacatatacaatt gagactggatggatatatgatatggaaaatgaaagtgataaatattatttcaatccagaaactaaaaa agcatgcaaaggtattaatttaattgatgatataaaatattattttgatgagaagggcataatgagaac gggtcttatatcatttgaaaataataattattactttaatgagaatggtgaaatgcaatttggttatataa atatagaagataagatgttctattttggtgaagatggtgtcatgcagattggagtatttaatacaccag atggatttaaatactttgcacatcaaaatactttggatgagaattttgagggagaatcaataaactata ctggttggttagatttagatgaaaagagatattattttacagatgaatatattgcagcaactggttcagt tattattgatggtgaggagtattattttgatcctgatacagctcaattagtgattagtgaatag

Antibody Preparation

In one embodiment, the present invention provides for a method for making a hybridoma that expresses an antibody that specifically binds to toxin B of C. difficile. The method contains the following steps: immunizing an animal with a composition that includes inactivated toxin B (e.g., toxoid B); isolating splenocytes from the animal; generating hybridomas from the splenocytes; and selecting a hybridoma that produces an antibody that specifically binds to toxin B. Kohler and Milstein, Nature, 256: 495, 1975. Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988.

Toxins can be inactivated, for example, by treatment with formaldehyde, glutaraldehyde, UDP-dialdehyde, peroxide, oxygen or by mutation (e.g., using recombinant methods). Relyveld et al., Methods in Enzymology, 93:24, 1983. Woodrow and Levine, eds., New Generation Vaccines, Marcel Dekker, Inc., New York, 1990. Genth et al., Inf. and Immun., 68(3):1094-1101, 2000. Mutant C. difficile toxins with reduced toxicity can be produced using recombinant methods. U.S. Pat. Nos. 5,085,862; 5,221,618; 5,244,657; 5,332,583; 5,358,868; and 5,433,945. A full-length or fragment of the toxins or toxoids can be used as immunogens.

In one embodiment, inactivated toxin B is used to immunize mice intraperitoneally or intravenously. One or more boosts may or may not be given. The titers of the antibodies in the plasma can be monitored by, e.g., ELISA (enzyme-linked immunosorbent assay) or flow cytometry. Mice with sufficient titers of anti-toxin B antibodies are used for fusions. Mice may or may not be boosted with the antigen 3 days before sacrifice and removal of the spleen. The mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line. The resulting hybridomas are then screened for the production of antigen-specific antibodies. Cells are plated, and then incubated in selective medium. Supernatants from individual wells are then screened by ELISA for human anti-toxin monoclonal antibodies. The antibody secreting hybridomas are replated, screened again, and if still positive for anti-toxin monoclonal antibodies, can be subcloned by limiting dilution. For example, the hybridoma clones CAN46G4, CAN46G13, CAN46G13a and CAN46G19 of the present invention have been subcloned. The subclones include, e.g., CAN46G4-1-2, CAN46G13-1-5, CAN46G13-1-8 and CAN46G19-3-2. The hybridomas CAN46G13-1-8, CAN46G4-1-2, CAN46G13-1-5 and CAN46G19-3-2 have been deposited with the American Type Culture Collection, Manassas, Va. (deposited on Aug. 23, 2012). The ATCC Patent Deposit Designation for each of the hybridomas is as follows: PTA-13257 (CAN46G13-1-8), PTA-13258 (CAN46G4-1-2), PTA-13259 (CAN46G19-3-2) and PTA-13260 (CAN46G13-1-5).

Adjuvants that may be used to increase the immunogenicity of one or more of the Clostridium difficile toxin antigens, particularly toxin B include any compound or compounds that act to increase an immune response to peptides or combination of peptides. Non-limiting examples of adjuvants include alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan trioleate (Span 85)), CpG-containing nucleic acid, QS21 (saponin adjuvant), MPL (Monophosphoryl Lipid A), 3DMPL (3-O-deacylated MPL), extracts from Aquilla, ISCOMS (see, e.g., Sjolander et al. (1998) J. Leukocyte Biol. 64:713; WO90/03184; WO96/11711; WO 00/48630; WO98/36772; WO00/41720; WO06/134423 and WO07/026190), LT/CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, Freund's, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

The immunized animal can be any animal that is capable of producing recoverable antibodies when administered an immunogen, such as, but not limited to, mice, rabbits, rats, hamsters, goats, horses, monkeys, baboons and humans. The host may be transgenic, but produce human antibodies/U.S. Pat. Nos. 8,236,311; 7,625,559 and 5,770,429, the disclosure of each of which is incorporated herein by reference in its entirety. Lonberg et al., Nature 368(6474): 856-859, 1994. Lonberg, N., Handbook of Experimental Pharmacology 113:49-101, 1994. Lonberg, N. and Huszar, D., Intern. Rev. Immunol., 13: 65-93, 1995. Harding, F. and Lonberg, N., Ann. N.Y. Acad. Sci., 764:536-546, 1995.

Antibody Assays

After the host is immunized and the antibodies are produced, the antibodies are assayed to confirm that they are specific for the antigen of interest and to determine whether they exhibit any cross reactivity with other antigens. One method of conducting such assays is a sera screen assay as described in U.S. Patent Publication No. 2004/0126829. Anti-toxin antibodies can be characterized for binding to the toxin by a variety of known techniques. For example, in an ELISA, microtiter plates are coated with the toxin or toxoid antigen in PBS, and then blocked with irrelevant proteins such as bovine serum albumin (BSA) diluted in PBS. Dilutions of plasma from toxin-immunized mice are added to each well and incubated. The plates are washed and then incubated with a secondary antibody conjugated to an enzyme (e.g., alkaline phosphatase). After washing, the plates are developed with the enzyme's substrate (e.g., ABTS), and analyzed at a specific OD. In other embodiments, to determine if the selected monoclonal antibodies bind to unique epitopes, the antibody can be biotinylated which can then be detected with a streptavidin labeled probe. Anti-toxin antibodies can be tested for reactivity with the toxin by Western blotting.

Neutralization assays can also be used to measure activity of the anti-toxin antibodies. For example, in vitro neutralization assays can be used to measure the ability of an antibody to inhibit a cytopathic effect on cells in culture (see Example 7 below). In one embodiment, in an in vitro neutralization assay, the present antibody, or antigen-binding portion thereof, at a concentration ranging from about 0.01 μM to about 50 μM, from about 0.2 μM to about 40 μM, from about 0.6 μM to about 30 μM, from about 2 μM to about 20 μM, from about 4 μM to about 10 μM, from about 0.2 μM to about 7 μM, from about 0.2 μM to about 10 μM, from about 4 μM to about 7 μM, from about 5 μM to about 15 μM, about 10 μM, about 0.01 μg/ml to about 200 μg/ml, about 0.01 μg/ml to about 150 μg/ml, about 0.01 μg/ml to about 100 μg/ml, about 0.01 μg/ml to about 50 μg/ml, about 0.01 μg/ml to about 25 μg/ml, about 0.01 μg/ml to about 10 μg/ml, about 0.01 μg/ml to about 5 μg/ml, about 0.1 μg/ml to about 2 μg/ml, about 1 μg/ml to about 2 μg/ml, about 0.5 μg/ml to about 2 μg/ml, about 0.25 μg/ml to about 2 μg/ml, about 0.1 μg/ml to about 2 μg/ml, about 0.06 μg/ml to about 2 μg/ml, or about 0.03 μg/ml to about 2 μg/ml, neutralizes a percentage of about 5 ng/ml, about 200 pg/ml, about 250 pg/ml, or about 200-250 pg/ml C. difficile toxin B. The percentages of toxin B neutralized by the present antibody, or antigen-binding portion thereof, may be greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99%.

In vivo assays can be used to measure toxin neutralization as well. In another embodiment, in an in vivo toxin B challenge experiment (e.g., procedures as described in Example 8, as well as Babcock et al., Human Monoclonal Antibodies Directed against Toxins A and B prevent Clostridium difficile-Induced Mortality in Hamsters. Infection and Immunity (2006) 74(11):6339), when the antibody, or an antigen-binding portion thereof, is administered to a mammal at a dosage ranging from about 1 mg/kg body weight to about 50 mg/kg body weight, from about 2 mg/kg body weight to about 40 mg/kg body weight, from about 3 mg/kg body weight to about 30 mg/kg body weight, from about 5 mg/kg body weight to about 20 mg/kg body weight, from about 8 mg/kg body weight to about 13 mg/kg body weight, or about 10 mg/kg body weight about 24 hours before the mammal is exposed to greater than about 75 ng, or about 75 ng of C. difficile toxin B, the mammal has a chance to survive. The chance of survival for the mammal may be greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99% within about 7 days or within about 4 days.

Hybridomas that produce antibodies that bind, preferably with high affinity, to the toxin can than be subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can then be chosen for making a cell bank, and for antibody purification.

To purify the anti-toxin antibodies, supernatants from the cultured hybridomas can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.).

Antibodies, or antigen-binding fragments, variants or derivatives thereof of the present disclosure can also be described or specified in terms of their binding affinity to an antigen. The affinity of an antibody for an antigen can be determined experimentally using any suitable method (see, e.g., Berzofsky et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and methods described herein). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., K_(D), K_(a), K_(d)) are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.

Pharmaceutical Compositions

The present invention also provides compositions containing an antibody or antigen-binding portion thereof described herein, and a pharmaceutically acceptable carrier. The composition may contain an isolated nucleic acid encoding the present antibody or antigen-binding portion thereof, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the composition is effective to reduce, eliminate, or prevent Clostridium difficile bacterial infection in a subject.

The invention also features methods of treating C. difficile disease in a subject by administering to the subject the present antibody or antigen-binding portion thereof in an amount effective to inhibit C. difficile disease. Routes of administration of the present compositions include, but are not limited to, intravenous, intramuscular, subcutaneous, oral, topical, subcutaneous, intradermal, transdermal, subdermal, parenteral, rectal, spinal, or epidermal administration.

The compositions of the present invention can be prepared as injectables, either as liquid solutions or suspensions, or as solid forms which are suitable for solution or suspension in liquid vehicles prior to injection. The composition can also be prepared in solid form, emulsified or the active ingredient encapsulated in liposome vehicles or other particulate carriers used for sustained delivery. For example, the composition can be in the form of an oil emulsion, water-in-oil emulsion, water-in-oil-in-water emulsion, site-specific emulsion, long-residence emulsion, stickyemulsion, microemulsion, nanoemulsion, liposome, microparticle, microsphere, nanosphere, nanoparticle and various natural or synthetic polymers, such as nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures, that allow for sustained release of the vaccine.

The present antibodies or antigen-binding portion are formulated into compositions for delivery to a mammalian subject. The composition is administered alone, and/or mixed with a pharmaceutically acceptable vehicle or excipient. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, the vehicle can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants. The compositions of the present invention can also include ancillary substances, such as pharmacological agents, cytokines, or other biological response modifiers. Methods of preparing the formulations are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 21st edition.

Compositions can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period appropriate to the age, weight and condition of the subject, the particular composition used, and the route of administration.

In one embodiment, a single dose of the composition according to the invention is administered. In other embodiments, multiple doses are administered. The frequency of administration can vary depending on any of a variety of factors, e.g., severity of the symptoms, degree of immunoprotection desired, whether the composition is used for prophylactic or curative purposes, etc. For example, in one embodiment, the composition according to the invention is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

The duration of administration of a polypeptide according to the invention, e.g., the period of time over which the composition is administered, can vary, depending on any of a variety of factors, e.g., subject response, etc. For example, the composition can be administered over a period of time ranging from about 10 minutes to about 1 day, from about 30 minutes to about 20 hours, from about 1 hour to about 15 hours, from about 2 hours to about 10 hours, from about 3 hours to about 8 hours, from about 4 hours to about 6 hours, from about 1 day to about 1 week, from about 2 weeks to about 4 weeks, from about 1 month to about 2 months, from about 2 months to about 4 months, from about 4 months to about 6 months, from about 6 months to about 8 months, from about 8 months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.

The present antibodies or antigen-binding portion thereof can be combined with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts to, e.g., stabilize, or increase or decrease the absorption or clearance rates of the present antibodies or antigen-binding portion thereof. Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, detergents, liposomal carriers, or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives. See e.g., the 21st edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. (“Remington's”).

In one aspect, the present antibodies or antigen-binding portion thereof are dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier. Examples of aqueous solutions include, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions and the like. The formulations can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. Additives can also include additional active ingredients such as bactericidal agents, or stabilizers. For example, the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate.

Solid formulations can be used in the present invention. They can be formulated as, e.g., pills, tablets, powders or capsules. For solid compositions, conventional solid carriers can be used which include, e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. Suitable pharmaceutical excipients include e.g., starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol.

For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories. Sayani, Crit. Rev. Ther. Drug Carrier Syst. 13: 85-184, 1996. For topical, transdermal administration, the agents are formulated into ointments, creams, salves, powders and gels. Transdermal delivery systems can also include, e.g., patches.

The present compositions can also be administered in sustained delivery or sustained release mechanisms. For example, biodegradeable microspheres or capsules or other biodegradeable polymer configurations capable of sustained delivery of a peptide can be included in the formulations of the invention (see, e.g., Putney, Nat. Biotechnol. 16: 153-157, 1998).

For inhalation, the present compositions can be delivered using any system known in the art, including dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like. Patton, Biotechniques 16: 141-143, 1998. Also can be used in the present invention are product and inhalation delivery systems for polypeptide macromolecules by, e.g., Dura Pharmaceuticals (San Diego, Calif.), Aradigrn (Hayward, Calif.), Aerogen (Santa Clara, Calif.), Inhale Therapeutic Systems (San Carlos, Calif.), and the like. For example, the pharmaceutical formulation can be administered in the form of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another aspect, the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes. Other liquid delivery systems include, e.g., air jet nebulizers.

Compositions or nucleic acids, polypeptides, or antibodies of the invention can be delivered alone or as pharmaceutical compositions by any means known in the art, e.g., systemically, regionally, or locally; by intra-arterial, intrathecal (IT), intravenous (IV), parenteral, intra-pleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa). Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in detail. Bai, J. Neuroimmunol. 80: 65-75, 1997. Warren, J. Neurol. Sci. 152: 31-38, 1997. Tonegawa, J. Exp. Med. 186: 507-515, 1997.

In one aspect, the pharmaceutical formulations comprising nucleic acids, polypeptides, or antibodies of the invention are incorporated in lipid monolayers or bilayers, e.g., liposomes. U.S. Pat. Nos. 6,110,490; 6,096,716; 5,283,185 and 5,279,833. Aspects of the invention also provide formulations in which nucleic acids, peptides or polypeptides of the invention have been attached to the surface of the monolayer or bilayer. For example, peptides can be attached to hydrazide-PEG-(distearoylphosphatidyl) ethanolamine-containing liposomes (see, e.g., Zalipsky, Bioconjug. Chem. 6: 705-708, 1995). Liposomes or any form of lipid membrane, such as planar lipid membranes or the cell membrane of an intact cell, e.g., a red blood cell, can be used. Liposomal formulations can be by any means, including administration intravenously, transdermally (see, e.g., Vutla, J. Pharm. Sci. 85: 5-8, 1996), transmucosally, or orally. The invention also provides pharmaceutical preparations in which the nucleic acid, peptides and/or polypeptides of the invention are incorporated within micelles and/or liposomes (see, e.g., Suntres, J. Pharm. Pharmacol. 46: 23-28, 1994; Woodle, Pharm. Res. 9: 260-265, 1992). Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art. Akimaru, Cytokines Mol. Ther. 1: 197-210, 1995. Alving, Immunol. Rev. 145: 5-31, 1995. Szoka, Ann. Rev. Biophys. Bioeng. 9: 467, 1980. U.S. Pat. Nos. 4,235,871; 4,501,728 and 4,837,028.

In one aspect, the compositions are prepared with carriers that will protect the peptide against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. U.S. Pat. No. 4,522,811.

It is advantageous to formulate parenteral or oral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. In one embodiment, the dosage of such compounds lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. In another embodiment, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Sonderstrup, Springer, Sem. Immunopathol. 25: 35-45, 2003. Nikula et al., Inhal. Toxicol. 4(12): 123-53, 2000.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antigen-binding portion of the invention is from about 0.001 to about 60 mg/kg body weight, about 0.01 to about 30 mg/kg body weight, about 0.01 to about 25 mg/kg body weight, about 0.5 to about 25 mg/kg body weight, about 0.1 to about 20 mg/kg body weight, about 10 to about 20 mg/kg body weight, about 0.75 to about 10 mg/kg body weight, about 1 to about 10 mg/kg body weight, about 2 to about 9 mg/kg body weight, about 1 to about 2 mg/kg body weight, about 3 to about 8 mg/kg body weight, about 4 to about 7 mg/kg body weight, about 5 to about 6 mg/kg body weight, about 8 to about 13 mg/kg body weight, about 8.3 to about 12.5 mg/kg body weight, about 4 to about 6 mg/kg body weight, about 4.2 to about 6.3 mg/kg body weight, about 1.6 to about 2.5 mg/kg body weight, about 2 to about 3 mg/kg body weight, or about 10 mg/kg body weight.

The composition is formulated to contain an effective amount of the present antibody or antigen-binding portion thereof, wherein the amount depends on the animal to be treated and the condition to be treated. In one embodiment, the present antibody or antigen-binding portion thereof is administered at a dose ranging from about 0.01 mg to about 10 g, from about 0.1 mg to about 9 g, from about 1 mg to about 8 g, from about 1 mg to about 7 g, from about 5 mg to about 6 g, from about 10 mg to about 5 g, from about 20 mg to about 1 g, from about 50 mg to about 800 mg, from about 100 mg to about 500 mg, from about 0.01 mg to about 10 g, from about 0.05 μg to about 1.5 mg, from about 10 μg to about 1 mg protein, from about 30 μg to about 500 μg, from about 40 pg to about 300 pg, from about 0.1 μg to about 200 mg, from about 0.1 μg to about 5 μg, from about 5 μg to about 10 μg, from about 10 μg to about 25 μg, from about 25 μg to about 50 μg, from about 50 μg to about 100 μg, from about 100 μg to about 500 μg, from about 500 μg to about 1 mg, from about 1 mg to about 2 mg. The specific dose level for any particular subject depends upon a variety of factors including the activity of the specific peptide, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

In therapeutic applications, the present compositions are administered to a subject at risk for Clostridium difficile bacterial infection or suffering from active infection in an amount sufficient to at least partially arrest or prevent the condition or a disease and/or its complications.

Use of Antibodies

The present antibodies or antigen-binding portion have in vitro and in vivo therapeutic, prophylactic, and/or diagnostic utilities. For example, these antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, inhibit, prevent relapse, and/or diagnose C. difficile and disease associated with C. difficile.

The antibodies or antigen-binding portion can be used on cells in culture, e.g., in vitro or ex vivo. For example, cells can be cultured in vitro in culture medium and contacted by the anti-toxin antibody or fragment thereof. The methods can be performed on cells present in a subject, as part of an in vivo (e.g., therapeutic or prophylactic) protocol. For in vivo embodiments, the contacting step is effected in a subject and includes administering an anti-toxin antibody or portion thereof to the subject under conditions effective to permit binding of the antibody, or portion thereof, to a toxin (e.g., toxin B) expressed by C. difficile in the subject, e.g., in the gut.

The antibody or antigen-binding portion thereof can be administered alone or in combination with another therapeutic agent, e.g., a second monoclonal or polyclonal antibody or antigen-binding portion thereof. In one example, the antibody or antigen-binding portion thereof specifically binds to C. difficile toxin B is combined with an antibody (monoclonal or polyclonal) or antigen-binding portion thereof specifically binds to C. difficile toxin A. In another example, the second agent is an antibiotic, e.g., vancomycin, bacitracin or metronidazole. The antibodies can be used in combination with probiotic agents such as Saccharomyces boulardii. The antibodies can also be administered in combinations with a C. difficile vaccine, e.g., a toxoid vaccine.

The antibody or antigen-binding portion thereof can also be administered in combination with one or more additional therapeutic agents, e.g., a second and third monoclonal or polyclonal antibody or antigen-binding portion thereof. In one example, the antibody or antigen-binding portion thereof specifically binds to C. difficile toxin B is combined with an antibody (monoclonal or polyclonal) or antigen-binding portion thereof which specifically binds to C. difficile toxin A and another antibody (monoclonal or polyclonal) or antigen-binding portion thereof which specifically binds to a different region of the C. difficile toxin B from the first C. difficile Toxin B antibody. In another example, the second or third monoclonal or polyclonal antibody or antigen-binding portion thereof is specific against binary toxin, or C difficile spore. In another example, the second agent is an antibiotic, e.g., vancomycin, bacitracin or metronidazole. In another example, the second agent is an antiparasitic, e.g. nitrazoxanide. The antibodies can be used in combination with probiotic agents such as Saccharomyces boulardii. The antibodies can also be administered in combinations with a C. difficile vaccine, e.g., a toxoid vaccine. In yet another use, the antibody or antigen-binding portion thereof can also be administered in combination with fecal transplants.

An anti-toxin antibody (e.g., monoclonal antibody) can also be used to isolate toxins by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-toxin antibody can be used to detect the toxin, e.g., to screen samples (e.g., in a stool sample, blood sample, culture sample, food samples) for the presence of C. difficile. Anti-toxin antibodies can be used diagnostically to monitor levels of the toxin in tissue as part of a clinical testing procedure to, for example, determine the efficacy of a given treatment regimen.

Vaccines

The present invention further encompasses vaccines and immunogen-containing compositions. The vaccines or immunogen-containing compositions may comprise one or more epitope recognized and/or bound by one or more of the present antibodies or antigen-binding portion thereof. In one embodiment, the vaccines or immunogen-containing compositions comprises one or more epitope recognized and/or bound by one or more of CAN46G4, CAN46G13, CAN46G13a, CAN46G19, CAN46G24, CAN33G1, antigen-binding portion of any of these antibodies, humanized form of any of these antibodies, or chimeric form of any of these antibodies. The vaccines or immunogen-containing compositions may contain the epitope, or may contain a peptide or protein having the epitope. In one embodiment, the epitope-containing portions, fragments, or peptides are derived from toxin B. For example, the epitope-containing portions, fragments, or peptides of toxin B are derived from the toxin B protein by proteolytic cleavage (e.g., by enterokinase, caspase, etc.). The epitope-containing portions, fragments, or peptides may also be chemically or recombinantly synthesized.

Such epitope-containing portions, fragments, or peptides of the toxins, when administered in the form of a vaccine or immunogen to a subject infected with C. difficile or afflicted with C. difficile-associated disease, may elicit a humoral response in the subject, i.e., antibodies having specificities for toxin B, thereby allowing the subject to mount an immune response against the toxins and to neutralize, block, reduce, ameliorate, cure, or treat the C. difficile-associated disease, infection, or CDAD in the subject. Accordingly, another embodiment provides a method of neutralizing, blocking, reducing, ameliorating, curing, or treating C. difficile infection or a C. difficile-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of the above-described vaccine or immunogen. In an embodiment, the subject elicits a humoral response to toxin B of C. difficile, thereby neutralizing, blocking, reducing, ameliorating, curing, or treating C. difficile-associated disease, infection, or CDAD in the subject. In another embodiment, the subject elicits a cellular immune response to toxin B of C. difficile. In another embodiment, the subject elicits both a humoral and a cellular immune response to toxin B of C. difficile. International Patent Publication No. WO2011130650.

Kits

The invention also provides kits containing an anti-toxin antibody or antigen-binding portion thereof. Additional components of the kits may include one or more of the following: instructions for use; another therapeutic agent, an agent useful for coupling an antibody to a label or therapeutic agent, other reagents, or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

Various combinations of antibodies can be packaged together. For example, a kit can include antibodies that bind to toxin B and antibodies that bind to toxin A (e.g., monoclonal anti-toxin A antibodies, or polyclonal antisera reactive with toxin A). The antibodies can be mixed together, or packaged separately within the kit.

Instructions for use can include instructions for therapeutic application including suggested dosages and/or modes of administration, e.g., in a patient with a symptom of CDAD. Other instructions can include instructions on coupling of the antibody to a label or a therapeutic agent, or for purification of a conjugated antibody, e.g., from unreacted conjugation components. The kits can be for diagnostic use, e.g., to detect the toxin, to screen samples (e.g., in a stool sample) for the presence of C. difficile. The kits can be used diagnostically to monitor levels of the toxin in tissue as part of a clinical testing procedure to, for example, determine the efficacy of a given treatment regimen.

The kit may or may not contain at least one nucleic acid encoding anti-toxin antibodies or fragment thereof, and instructions for expression of the nucleic acids. Other possible components of the kit include expression vectors and cells.

The present antibodies or antigen-binding portion, compositions and methods can be used in all vertebrates, e.g., mammals and non-mammals, including human, mice, rats, guinea pigs, hamsters, dogs, cats, cows, horses, goats, sheep, pigs, monkeys, apes, gorillas, chimpanzees, rabbits, ducks, geese, chickens, amphibians, reptiles and other animals.

The following examples of specific aspects for carrying out the present invention are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

EXAMPLES Example 1 Hybridoma Fusion

Methods and reagents for generating monoclonal antibodies are well known and encompass immunization protocols as well as techniques for isolating and fusing splenocytes. A classical hybridoma fusion was performed using the standard somatic cell hybridization technique of Kohler and Milstein, Nature, 256: 495, 1975. See generally, Harlow, E and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1988. In general, mice are immunized with inactivated toxin, or toxoids, fragments of the toxin, or whole toxin. Mice received their first immunization with antigen using Complete Freund's Adjuvant (CFA) or other adjuvants, followed by subsequent boosters every other week (up to a total of 4) with antigen and Incomplete Freund's Adjuvant (IFA). A trial bleed from the medial saphenous vein was performed, and the serum tested to check for titers of anti-toxin B antibody. If IgG titers were sufficient, fusions were performed using 1 or 2 mice at a time. Mice were given a final push intraperitoneally (IP) with a toxoid B/toxin B combination in PBS three days prior to the fusion.

On the day of the fusion, mice are sacrificed and their spleens removed. Splenocytes are washed from the spleen using a syringe and needle and collected in a 50 ml tube for fusion with myeloma cells. Myelomas are an immortal tumor cell line used as fusion partners, grown in the presence of 8-azaguanine(8-aza), a toxic nucleotide analog which blocks the salvage pathway. Cells grown in the presence of 8-aza survive only by incurring defective mutations in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene. B cells are fused with the myeloma cells using Polyethylene Glycol 1500. Fused cells are mixed into semi-solid agarose with drug selection and plated out into petri dishes. HAT media containing Hypoxanthine, Aminopterin, and Thymidine is used for drug selection. Aminopterin is a drug which inhibits the de novo pathway for nucleotide metabolism which is absolutely required for survival/cell growth in myeloma lines defective in HGPRT, and allows selection usually within 24-48 hours.

Example 2 Hybridoma Screening

The next step is screening of the growing hybridomas. A commercial semisolid agarose within which the cells grow as a mass of cells in the 3-D matrix was used. This facilitates picking clusters by hand (by visual inspection) and transferring these clonal clusters into a 96 well plate containing suitable media. The cells were allowed to grow for 3-7 days and then the supernatant removed for screening and replaced with fresh media. Positive binding in ELISA (or other tests) resulted in continuing to grow the hybridomas by transferring them into larger tissue culture vessels with increasing volume. The mAbs were isotyped using a suitable commercial isotyping kit for murine mAbs using the spent supernatant. The decision to move a clone to the next stage of selection is based on its reactivity to native toxin B using an ELISA and its survival, usually based upon serial dilutions and reactivity of at least ⅛ or 1/16 or higher, as well as IgG class; therefore the number of clones decreased throughout the selection procedure. The murine mAbs that underwent further characterization were: CAN33G1, CAN46G4, CAN46G13, CAN46G13a, CAN46G19 and CAN46G24.

Example 3 ELISA Assay of Mouse Monoclonal Antibodies

An ELISA was used to test the binding of the mAbs against whole toxin B and recombinant toxin B fragments 1 and 4 as well as to determine if they were cross-reactive to whole toxin A. The mAb clones were compared to purified anti-toxoid B mouse pAb (polyclonal Ab). The ELISA plate was coated with 100 ng of toxin B fragment 1, fragment 4, or 400 ng of whole toxin B so that the coatings were equimolar. The wells were blocked with 5% skim milk then probed with serially diluted CAN46 series mAbs (0.1 μg/ml to 1 μg/ml) and binding was detected with a commercial goat anti-mouse IgG-HRP antibody. Negative and positive controls were also run. The polyclonal toxoid B antibody (pAb) served as the positive control, and is derived from immunized mice. The murine anti-toxin A mAb CAN20G2 is specific for Toxin A and was used as the negative control. The secondary antibody control is for the murine secondary antibody. The plate was read at 405 nm after 15 min incubation with substrate. The titration data for each antibody is shown in FIG. 1.

Results:

As shown in FIG. 1, CAN46 series mAbs bind to whole toxin B and toxin B fragments at a similar level to the mouse pAb, with the exception of reduced binding with CAN33G1. CAN46G4, CAN46G19 and CAN46G24 bind to toxin B fragment 4 at a similar level to the mouse pAb. CAN46G13a binds to toxin B fragment 1 at a similar level to the mouse pAb. None of the CAN46 mAbs showed cross-reactivity to toxin A.

Example 4 Competition ELISA Assay of Mouse Monoclonal Antibodies

An ELISA was performed to test if the CAN33 or CAN46 mAbs compete with MDX 1388 (Medarex, US Patent Publication No. US 2005/0287150 A1) or hPA-41 (Progenics Pharmaceuticals, Inc., International Patent Publication No. WO 2011/130650; Marozsan et al., Protection Against Clostridium difficile Infection With Broadly Neutralizing Antitoxin Monoclonal Antibodies, J. Infect. Dis. 2012 September; 206(5):706-13) for binding to Toxin B. The ELISA plate was coated with 400 ng of whole Toxin B. The wells were blocked with 5% skim milk and then probed with the mAb mixtures as follows: the murine CAN33 and CAN46 mAbs were prepared at 1 μg/ml and serially diluted two-fold. In order to provide a baseline OD of approximately 1.0, a dilution of 1/1,650,000 was prepared for MDX1388 anti-toxin B mAb used as a control, dilution based on previous data generated in house and mixed with the CAN33 and CAN46 mAbs 1:1. Similarly, hPA-41 was diluted to 1/335,000 and mixed 1:1 with the serially diluted CAN33 and CAN46 mAbs as above. Binding for CAN33 and CAN46 mAbs was detected with a commercial goat anti-mouse IgG-HRP antibody. Binding for MDX1388 and hPA-41 was detected with a commercial goat anti-human IgG-HRP antibody. Negative and positive controls were also run. Excess Toxin B mixed 1:1 with either MDX1388 or hPA-41 served as the positive control. MDX1388 and hPA-41 were also diluted with just phosphate buffered saline for a negative control with no competitive mAb. The plate was read at 405 nm after 15 min incubation with substrate.

Results:

As shown in FIGS. 2 and 3, CAN33 and CAN46 mAbs do not compete with MDX-13-88 or hPA-41 for binding to whole toxin B. All of the mAbs show similar binding patterns to those for the negative control containing no competitive mAb. None of the mAbs showed competition with either MDX-1388 or hPA-41 indicating they bind different epitopes on whole toxin B.

Example 5 Western Blot of Mouse Monoclonal Antibodies

A 4-12% gradient SDS-PAGE gel was run for 1.5 hours at 200 volts with a combination of C. difficile proteins: recombinant toxin B fragment 1, (82 kDa), recombinant toxin B fragment 4 (85 kDa), whole toxin B (280 kDa), and commercial BSA. The gel was then transferred to a nitrocellulose membrane for 1 hour 15 min at 45 volts. The membrane was blocked overnight at 4° C. with 5% skim milk in 1×TBST. The next day the mAbs (1° Ab) were diluted in 2.5% skim milk in 1×TBST at concentrations ranging from 2 μg/ml to 5 μg/ml depending on the antibody and used to probe the membrane containing the transferred products for 2 hours at room temperature (RT) on a shaker. The membranes were then washed with 1×TBST to remove unbound 1° Ab and probed with anti-mouse IgG-HRP (2° Ab) at a dilution of 1:4000 to 1:5000 for 1.5 hours at RT on a shaker.

Results:

As shown in FIGS. 4, 5 and 6, CAN46G4, CAN46G13, CAN46G19 and CAN46G24 showed binding to recombinant toxin B fragment 4 and whole toxin B. CAN46G13a showed binding to recombinant toxin B fragment 1 and whole toxin B. They all showed no cross-reactivity to the negative control (BSA).

Example 6 Affinity Analysis of Mouse Monoclonal Antibodies

Biolayer interferometry was used to measure the interactions between whole toxin B and the anti-toxin B antibodies. The Octet QKe instrument (ForteBio) was equipped with Streptavidin (SA) biosensors. 40 μg/ml of biotinylated antibody was coupled to SA sensors and toxin B, in a dilution series from 50 nM to 0.78 nM, were reacted on the antibody-coated pins followed by a dissociation step in PBS-Triton. The results were then analyzed using ForteBio Data Analysis software to determine the dissociation constant (K_(D)), which is the measure used to describe the binding strength between antibody and antigen, k_(on)(1/Ms), the on-rate at which antibody antigen complexes form, and k_(dis)(1/s), the off-rate at which the antibody antigen complexes dissociate. Table 4 shows affinity (equilibrium dissociation constant (K_(D)) ratio of k_(dis)/k_(on) between antibody and antigen is inversely related to affinity whereby the lower the K_(D) the higher the affinity) of purified murine CAN33 and CAN46 mAbs.

TABLE 4 Affinity data for CAN46G and CAN33G versions, hPA-41 and MDX1388 Antibody K_(D) (M) k_(on)(1/Ms) k_(dis)(1/s) CAN46G4 1.41E−09 7.18E+04 1.01E−04 CAN46G13 1.17E−09 1.28E+05 1.49E−04 CAN46G13a 8.57E−09 8.08E+05 6.93E−03 CAN46G19 1.27E−09 1.14E+05 1.45E−04 CAN46G24 1.89E−09 1.49E+05 2.80E−04 hPA-41 9.83E−11 5.38E+05 5.30E−05 CAN33G1 1.09E−08 5.44E+04 5.91E−04 MDX1388 3.84E−09 3.62E+04 1.39E−04

Example 7 Epitope Binning of Mouse Monoclonal Antibodies

The Octet QKe is a label free real-time biosensor that uses disposable fiber-optic sensors that detect biomolecular interactions via biolayer interferometry. The epitope binning assay was performed against the previously characterized MDX1388 mAb to examine whether the present toxin B mAbs share a similar or a different epitope with MDX1388. Secondly, the assay was used to confirm shared single or potentially multiple epitope bins between the toxin B mAbs. The classical sandwich method was used and involves coupling the mAb to sensor, binding antigen, and then binding to another mAb. The second mAb can bind the captured Ag only if its epitope does not overlap that of the immobilized mAb. For example, biotinylated CAN46G24 antibody is coupled to a Streptavidin (SA) biosensor. The bound antibody is then incubated with free Toxin B and free CAN46G24. The CAN46G24-toxin B complex is again incubated with free antibody. A large nm shift in wavelength indicates binding of the analyte indicating that CAN46G24 and the free antibody have different epitopes. 1 Biotinylated CAN46G24 to SA biosensors; 2 Free whole toxin B forming a complex with CAN46G24; 3 Free CAN46G24 associating with biotinylated CAN46G24-Toxin B complex; 4 Association sample curves; 5 Dissociation step.

Results:

In FIG. 7, the nm shift for both the MDX1388 and CAN46G13a samples indicate binding to an exposed and distinct epitope. There is no nm shift for CAN46G4 and CAN46G24 samples indicating shared or spatially related epitopes to CAN46G19.

Example 8 Clostridium difficile Toxin B Neutralization Assay with HT-29 Cells (Human Colon Carcinoma Epithelial Cells) Using the xCELLigence™ Platform Cell Line

The HT-29 cells are an adherent human colon carcinoma epithelial cell line. These cells have been selected since they represent a relevant in vitro model to infection with TcdB.

xCELLigence™ Platform

The xCELLigence™ is a real-time label-free cell analysis (RTCA) system based on an electronic impedance cell sensing measurement that evaluates changes in cell characteristics in real-time. Cell growth and cytotoxicity can be detected by monitoring the increase or decrease of a dimensionless parameter called cell index (CI). When adherent cells are cultured within the custom 96-well plate, cell growth characteristics can be monitored in real-time by changes in electrical impedance as measured by the gold electrodes embedded within each well.

The CI measurement is based upon four parameters: 1) cell number, 2) cell size and morphology, 3) cell viability, and 4) cell adhesion. An increase in any one of these parameters leads to an increase in the CI. Conversely, a decrease in any one of these parameters leads to a decrease in CI.

Procedure

HT-29 cells were trypsinized from a T-75 flask and added to a Roche 96-well E-plate® at 8000 cells/well, and incubated about 4 hours at 37° C. During the 4-hr incubation, sample dilutions were prepared on a 96-well U-bottom plate. Samples were then overlayed with an appropriate dilution of TcdB (0.5-50 ng/mL range, dilution dependant on toxin lot). The plate is then incubated at 37° C. for about 60 minutes. After completion of initial cell incubation, the cells were overlayed with the toxin/sample preparation then incubated for a minimum of 72 hours at 37° C. Impedance measurements were taken every 30 minutes throughout the incubation period. This data is plotted in real-time using the xCELLigence™ RTCA software. A single time point representing the optimal time point (either for toxin cytotoxicity or neutralization) was selected. The data from that single time point is used to create a 4-parameter logistic (4-PL) curve for analysis. If sample potency is being determined, the sample curves are constrained against the “reference” sample. Curve constraint is used to constrain the upper/lower asypmtotes, and slope of the curve. This allows for each curve to shift horizontally along the x-axis based upon the curves IC₅₀ value. For potency determination the IC₅₀ value of the standard is divided by the IC50 value of the sample.

Initial Evaluation for TcdB Cytopathogenicity Detection with xCELLigence

Toxin B cytopathogenicity on HT-29 cells was first evaluated to determine the suitability of the xCELLigence™ platform. FIG. 8 shows the dose response curve of TcdB at 2-fold dilutions, demonstrating that increasing concentrations of Tcd B on HT-29 cells leads to cytopathogenicity as indicated by a decrease in the cell index. The increasing cell index with decreasing concentrations of TcdB demonstrates a suitable detection of cytopathogenicity. A TcdB concentration of 5 ng/mL is showing approximately full cytopathic effect and indicates this as a suitable concentration to evaluate toxin neutralization.

Cell attachment Phase—xCelligence™ Method

This phase included the following steps. (1) Trypsinized cells in source flask. (2) Added 2 mL of trypsin to flask and washed cells to remove traces of media then aspirated. (3) Added 3 mL of trypsin and incubated at 37° C. for approximately 8 minutes. (4) Added 6 mL of assay media to flask. (4) Centrifuged suspended cells at 800 rpm for 7 minutes. (5) Aspirated supernatant and resuspended cells with 6 mL of assay media. (6) Counted cells and calculated required volume of cells for plating at 8000 cells/well. (7) To a 96 well E-plate added 100 μL of assay media to all wells. (8) Performed background reading on xCelligence. (10) Added 50 μL of 1.0×10⁶ cells/mL suspension to these wells for a final 8000 cells/well seeding density. (11) (15) Incubated plate at room temperature for 20-30 minutes to allow cells to settle evenly. (16) Placed plate in 37° C. incubator with 5% CO₂ overlay 4-5 hours.

Toxin B Preparation:

(1) Prepared Toxin B Overlay by Diluting Primary Stock (409.6 μg/mL) to 5 ng/mL (2) Prepared Toxin B for titration by diluting primary stock to 80 ng/mL. (3) Dilutions of primary stock were performed as shown in Table 5.

TABLE 5 TcdB Test Volume of Volume of 10% Sample Concentration TcdB (μL) Medium (μL) Toxin Overlay (i) 500 ng/mL 12.2 9988.8 (Stock = (ii) 5.0 ng/mL 120 of (i) 11,880 409.6 μg/mL) Toxin titration     80 ng/mL 160 of (i) 840

Sample Preparation:

To test potency, all the monoclonal antibodies were prepared at appropriate concentrations as shown in Table 6.

TABLE 6 Sample Test Volume of Volume of 10% Sample Concentration TcdB (μL) Medium (μL) MDX1388 (Standard; 30 μg/mL 10 690 Medarex anti-TcdB) 2.1 mg/mL hPA-41.1 (5.3 mg/mL) (i)1000 μg/mL 10 43 (Progenics anti-TcdB) (ii) 10 μg/mL  10 of (i) 990 S1 = CAN46G4-1-2 (i)100 μg/mL 10 850 (8.6 mg/mL) S2 = CAN46G19-3-2 (i)100 μg/mL 10 530 S3 = CAN46G13-1-5 (i)1000 μg/mL 10 165 (17.5 mg/mL) (ii) 300 μg/mL 150 of (i) 350 S4 = CAN46G24-2-3 300 μg/mL 30 780 (2.7 mg/mL) S5 = CAN46G13-1-8 (i)1000 μg/mL 10 194 (20.4 mg/mL) (ii) 300 μg/mL 150 of (i) 350 S6 = CAN46G13-1 (i) 1000 μg/mL 10 42 (5.4 mg/mL) (ii) 30 μg/mL  15 of (i) 450

Dilution Plate Preparation-xCelligence

The following was performed using a U-bottom 96-well plate: (1) Added 112.5 μL of assay media to wells B2-H11, and E12-H12. (2) Added 225 μL of media to wells A12-D12. (3) Added 100 μL of assay media to wells B1-H1. (4) Added 150 μL of sample to corresponding wells as shown below in Table 7. (5) Serially diluted each sample 4-fold by transferring 37.5 μL from Row A and adding to Row B, mixed and repeated down through to Row H. (6) Serially diluted the toxin titration wells 3-fold by transferring 50 μL from row A to row B, mixing, then continuing to serially dilute through to row H. (7) Samples and Toxin Control (TC) were overlayed with 112.5 μL of Toxin B (5 μg/mL). (8) The toxin titration wells were overlayed with 100 μL of assay media. (9) Plate(s) was shaken on a plate shaker until homogeneous. (10) Incubated at 37° C. with 5% CO₂ for 60-90 minutes. Table 7 shows the xCelligence dilution plate layout.

TABLE 7 xCelligence Dilution Plate Layout 1 2 3 4 5 6 7 8 9 10 11 12 A Toxin B Standard Internal Sam- Sam- Sam- Standard Internal Sam- Sam- Sam- CC B Titration (MDX1388) Control ple 1 ple 2 ple 3 (MDX1388) Control ple 1 ple 2 ple 3 C Standard Standard D (hPA-41.1) (hPA-41.1) E TC F G H CC = Cell control (8000 cells/well); TC = Toxin control (5 ng/mL)

Sample Addition to Cell Plates:

(1) Following completion of incubations, the cell and dilution plates were removed from incubator. (2) (3) Transferred 50 μL of samples from dilution plate to appropriate wells of cell plate. (4) Incubated 72 hours at 37° C. with a 5% CO₂ overlay.

Data Analysis:

(1) Plate data at the 72 hour time point was fit to a 4-parameter logistics (4-PL) curve for each individual sample using Softmax Pro (v.5.4) software. (2) Standard and sample curves were constrained (upper/lower asymptotes, and slope), and the IC₅₀ value of the standard was divided by the IC₅₀ of the sample to determine a potency estimate (when applicable).

The procedures of this Example were also performed on mAbs.

Results

The murine CAN46 mAbs show similar EC₅₀ values to MDX-1388, with CAN46G24 demonstrating an even greater level of neutralization in vitro.

Table 8 summarizes the EC₅₀ data for each mAb demonstrating that CAN46G24 and CAN46G13 are the most neutralizing of the clones when compared to MDX1388.

TABLE 8 Mean EC₅₀ Sample (μg/mL)¹ n = Progenics hPA-41 18.3 6 CAN46G24 44.2 2 Medarex MDX1388 125.4 6 CAN46G13 136.0 2 CAN46G19 141.5 2 CAN46G4 142.5 2 ¹The EC50 value is the concentration of antibody which neutralizes 50% of the TcdB toxin dose.

Example 9 Clostridium difficile Toxin B In Vitro Neutralization Assays with HT-29 Cells

For the in vitro neutralization assays with HT-29 cells, the percent neutralization ranges in Table 9 were compiled from data from the murine antibodies. The concentration of toxin B used was 5 μg/ml.

TABLE 9 Antibody Concentration Neutralization % (μg/mL) of 5 ng/ml Toxin B 100 57.8-99.5% 25 34.1-102.6% 6.25 24.2-76.7% 1.56 14-71.9% 0.39 7.8-64.6% 0.1 0-53.3% 0.02 0-28.4% 0.01 0-20.4%

Example 10 Mouse In-Vivo Toxin Challenge

The mouse in vivo toxin challenge test was based on previous publications with some modifications (Babcock et al., Human Monoclonal Antibodies Directed against Toxins A and B prevent C. difficile-Induced Mortality in Hamsters, Infection and Immunity (2006)). Balb/c mice weighing 20-30 g were given 250 μg of mAb or controls at day 0 and allowed to rest. After 24 hrs, the mice were given a lethal dose of TcdB (75 ng). This dose kills 90-100% of animals by 24 hours in an unprotected state. The mice were observed for 4 days for signs of abnormality and local and systemic disease. All observations were recorded and the % survival was determined for each treatment group.

Results

As shown in FIG. 9, the study results show that the CAN46 mAbs protect mice against toxin B. All the Can46 mAbs, CAN46G4, CAN46G13, CAN46G13a, CAN46G19 and CAN46G24, were efficacious at the dose of 0.25 mg/mouse in protecting against lethal toxin B challenge with 100% survival 4 days after the toxin B challenge. CAN33G1 was not able to protect mice at a dose of 0.25 mg/mouse with 0% survival 4 days after the toxin B challenge.

Example 11 V Gene Sequencing

RNA was isolated from each of the CAN46G parental hybridoma clonal cell line using the RNeasy Mini Kit. The amplification of V genes from the RNA was performed using the Qiagen OneStep RT-PCR Kit. Several combinations of primer sets were used as follows: for immunoglobulin variable region gene sequence confirmation from the hybridomas, a set of Variable region gene (V-gene) subgroup-specific oligonucleotide primers are used. These could include 5′mVK-Lead-1, 3′KappaConstRT, 5′mVH-Lead-2, 5′mVH-Lead-2A, and 3′mIG1-2C RT. In order to rule out potential contamination from the known and endogenous aberrant kappa light chain V-gene mRNA (found within P3X63 myelomas) (Yuan, X. et al., J. Immunol. Methods, 294: 199-207 (2004)), the RT-PCR was also performed using non-subgroup specific primer sets that could include, 5′mVK-Lead-1A, 5′mVK-Lead-1A, 5′mVK-Lead-3, 5′mVK-Lead-3A, 5′mVH-IGHV1-Lead, 5′mVH-Lead-1, 5′mVH-Lead-3, 5′mVH-Lead-4, and 5′mVH-Lead-5. Refer to FIG. 10 for a list of the primers and their sequences. The primers use degenerate base symbols are IUPAC (International union of pure and applied chemistry) codes for representing degenerate nucleotide sequence patterns.

The results of the PCR amplification reactions were determined by examining the PCR products on an analytical agarose gel, and the visualized bands at approximately 300-500 bp were gel isolated for cloning. The extracted DNA was directly TA cloned into the pCR2.1-TOPO vector using the low melt agarose method in the TOPO TA Cloning manual. Each CAN46G clone reactions were sequenced in both directions using the M13 Forward and M13 Reverse primers. Sequence data was analyzed using DNAStar Lasergene software. FIG. 11 shows the resulting rearranged V-gene sequences compared to IMGT/V-Quest reference directory sets and to the NCBI immunoglobulin blast search. The figure includes results for both the VH and VL sequences of the murine parental clones CAN46G4, CAN46G13a, CAN46G19, CAN46G24, CAN46G13 and CAN33G1. Analysis of CAN46G24 and CAN46G13 revealed identical sequences for VH and VL sequences of the murine parental clones.

Example 12 Humanization of CAN46G

Three humanized IgG/k versions of each CAN46G mAb have been created. For the humanized versions, maximum identity alignment with human germline alleles was used (NCBI website) to help to identify acceptor frameworks. All 6 CDRs corresponding to heavy and light chains were inserted. Other residues were changed or maintained due to surface exposure or involvement in folding or interchain contacts, respectively. This resembles the “superhumanization” approach where CDR matching rather than total framework is used in a variation of the use of germline sequences as acceptor frameworks. In the case of Tan et al., J. Immunol.

2002, 169:1119-1125, the authors used the CDR sequences and tried to match the so called canonical classes of CDRs based upon the Kabat classification system. However, because particular CDRs are germline encoded and particular canonical conformations tend to be found in certain frameworks, the “Superhumanization” method of choosing acceptor frameworks does not in all cases result in the selection of a different candidate acceptor framework. It is empirical and remains to be tested for multiple mAb specificities. This is in part because the straight-up alignment of frameworks for identity inherently encompasses the CDRs as well in the comparison. cdrCAN46G CDR Grafted Only (FIG. 12)

The best matching human germline allele for both V_(H) and Vk were used as an acceptor framework for grafting the CDRs. No other changes were made to the acceptor frameworks.

huCAN46 and rehuCAN46 “Human Engineered” (FIGS. 13 and 14)

This humanized versions were generated using a strategy most similar to the “human engineering” strategy used by Studnicka et al. to humanize a murine mAb to CD5 (Studnicka et al, Human-engineered monoclonal antibodies retain full specific binding activity by preserving non-CDR complementarity-modulating residues, Protein Eng. 1994 June; 7(6):805-14). Essentially, the closest human germline allele for CAN46G mAbs V_(H) and Vk were identified, individually, and designed for use as acceptor frameworks. The rehuCAN46 mAbs were further resurfaced by substitution(s) made on surface exposed amino acids to correspond to the adopted human frameworks without disruption of the CDRs.

Example 13 ELISA Assay of Humanized Monoclonal Antibodies

An ELISA was used to test the binding of the mAbs against whole toxin B and toxin A to determine if they were cross-reactive to whole toxin A. The ELISA plate was coated with 400 ng of whole toxin B or toxin A so that coating was equimolar. The wells were blocked with 5% skim milk then probed with serially diluted CAN46 series mAbs (0.1 μg/ml to 1 μg/ml) and binding was detected with a commercial goat anti-human IgG-HRP antibody. Negative and positive controls were also run. The plate was read at 405 nm after 15 min incubation with substrate. The titration data for each antibody is shown in FIG. 40.

Results:

As shown in FIG. 37, humanized CAN46 mAbs expressed in CHOK1 SV cells from the CHO construct, have retained the binding characteristics of their respective mouse mAbs and bind to whole toxin B. Humanized CAN46G13a mAbs bind to toxin B at a reduced level compared to the other Cangene mAbs and control mAbs. None of the CAN46 mAbs showed cross-reactivity to toxin A.

Example 14 Western Blot of Humanized Monoclonal Antibodies

A 4-12% gradient SDS-PAGE gel was run for 1.0 hour at 200 volts with a combination of C. difficile proteins: whole toxin B, recombinant toxin B fragment 1, recombinant toxin B fragment 4, and whole toxin A. The gel was then transferred to a nitrocellulose membrane for 1 hour at 45 volts. The membrane was blocked for one hour at room temperature or overnight at 4° C. with 5% skim milk in 1×TBST. The mAbs (1° Ab) were diluted in 5% skim milk in 1×TBST at concentrations ranging from 0.038 μg/ml to 5 μg/ml depending on the antibody and used to probe the membrane containing the transferred products for 5 hours at room temperature (RT) or overnight at 4° C. on a shaker. The membranes were then washed with 1×TBST to remove unbound 1° Ab and probed with anti-human IgG-HRP (2° Ab) at a dilution of 1:4000 to 1:5000 for 1.5 hours at RT on a shaker.

Results:

As shown in FIGS. 34 and 35, humanized versions of CAN46G19 and CAN46G24 showed binding to recombinant toxin B fragment 4 (85 kDa) and whole toxin B (280 kDa). Humanized versions of CAN46G13a showed binding to recombinant toxin B fragment 1(82 kDa) and whole toxin B (280 kDa). None of the humanized CAN46 mAbs tested were cross reactive to Toxin A (308 kDa).

Example 15 In Vitro Neutralization Assay of Humanized Antibodies

An in vitro neutralization assay for C. difficile toxins using CT.26 cells was performed to test the neutralization capability of the humanized mAb variants against C. difficile toxin B. The CT.26 cells were seeded in a 96 well plate at a concentration of 2.5-3×10⁴ cells/100 μl/well and the plate was incubated in a 5% CO₂ incubator for 4-5 hours at 37° C. Two blank wells containing media (no cells) were also included in the plate.

Toxin and toxin/Ab stock solutions were prepared and diluted to the desired concentrations using Roswell Park Memorial Institute (RPMI) media. The diluted toxin controls and toxin/Ab test mixtures were incubated at room temperature for 1 hour. Thereafter, media was removed from the wells and incubated for 48 hours at 37° C. and 5% CO₂ in the presence of either media alone (Cell control), toxin alone (Toxin control), or toxin/Ab mixtures. Plates were returned to incubate for 48 hours at 37° C. and 5% CO₂. Next, WST-1 detection reagent was added to each well (10 μl of reagent/100 μl volume in the well) and incubated an additional hour at 37° C. and 5% CO₂ prior to shaking the plate for 1 minute and reading absorbance at 450 nm.

Cell viability was determined based on the cell controls as below:

% Cell viability=Mean OD of test/Mean OD of cell control×100.

Toxin neutralization is calculated by the formula as below:

% Neutralization=(Sample OD−Toxin control OD)/Cell control OD−Toxin control OD)×100.

Results

As shown in FIGS. 15 a and 15 b, the huCAN46G4, rehuCAN46G4, huCAN46G19 and rehuCAN46G19 provided the highest level of protection (neutralization) at all mAb concentrations. These levels of neutralization were comparable to the original murine versions. MDX1388 mAb also shows comparable neutralizing ability while the cdrCAN46G4 and cdrCAN46G19 both show much reduced or no activity when compared to the original murine version or control mAb MDX1388.

For the in vitro neutralization assays with CT.26 wt cells, the percent neutralization ranges in Table 10 were compiled from data from two humanized mAbs huCAN46G4 and huCAN46G19. The concentration of toxin B used was 200-250 pg/ml.

TABLE 10 Antibody % Concentration mAb Neutralization Range (μg/ml) huCan46G4 >20% 0.03125-2.0 >30% 0.0625-2.0 >40% 0.0625-2.0 >50% 0.125-2.0 >60% 0.25-2.0 >70% 0.25-2.0 >80% 0.5-2.0 >90% 1.0-2.0 huCan46G19 >20% 0.0156-2.0 >30% 0.0156-2.0 >40% 0.0156-2.0 >50% 0.0156-2.0 >60% 0.03125-2.0 >70% 0.03125-2.0 >80% 0.0625-2.0 >90% 0.0625-2.0 Furthermore, FIGS. 19-23 demonstrate the capacity of CAN46 mAbs purified from either HEK293F cells expressing the Per.C6-based construct (depicted as 293F in the figure legends) or CHOK1 SV cells expressing the CHO-based construct (depicted as CHO in the figure legends) in neutralizing Toxin B challenge against CT-26 cells. Specifically, huCAN46G24 mAbs in both CHO and Per.C6 constructs showed superior neutralization to the rehuCAN46G24 constructs (FIG. 19). In FIG. 20, the chimeric CAN46G13a mAb showed superior neutralization to the rehuCAN46G13a in CHO and the huCAN46G13a in both constructs. In FIG. 21, the huCAN46G19 mAbs in both constructs (CHO and HEK293F) showed superior neutralization to the rehuCAN46G19 constructs. In FIG. 22, the three mAbs, huCAN46G4, huCAN46G19 and huCAN46G24 showed comparable neutralization, with 100% neutralization between 1-2 ug/ml. In FIG. 23, the rehuCAN46G24 showed moderate neutralization of Toxin B and the huCAN46G13a and rehuCAN46G13a mAbs showed weak neutralization of Toxin B, in comparison to huCAN46G24. In FIG. 25, the huCAN46G24 mAb in CHO showed the highest neutralizing capability of the CHO humanized CAN46 mAbs tested, with 100% neutralization at about 0.18 ug/ml. The rehuCAN46G19 and rehuCAN46G24 in CHO showed moderate neutralization, with 100% neutralization at about 0.3 ug/ml. Taken together, these results demonstrate the CAN46 mAbs neutralize the cytotoxic effects of toxin B against CT26 cells in vitro using humanized monoclonal antibodies describe in the invention with specificity against either domain 1 or 4 of toxin B.

Example 16 Mouse In-Vivo Toxin Challenge

The mouse in vivo toxin challenge test was based on previous publications with some modifications (Babcock et al., Human Monoclonal Antibodies Directed against Toxins A and B prevent C. difficile-Induced Mortality in Hamsters, Infection and Immunity (2006)). Balb/c mice weighing 20-30 g were given 250 μg of mAb or controls at day 0 and allowed to rest. After 24 hrs, the mice were given a lethal dose of TcdB (75 ng). This dose kills 90-100% of animals by 24 hours in an unprotected state. The mice were observed for 4 days for signs of abnormality and local and systemic disease. All observations were recorded and the % survival was determined for each treatment group.

Results

As shown in FIG. 16-17, the study results show that the humanized versions of CAN46 mAbs (purified from HEK293F cells expressing the Per.C6 based construct) protect mice against toxin B challenge. FIG. 16 shows, that all the CAN46G13a humanized mAbs, huCAN46G13a (10%), cdrCAN46G13a (20%), rehuCAN46G13a (30%) were not efficacious at the dose of 0.25 mg/mouse in protecting against lethal toxin B challenge 3 days after the toxin B challenge. FIG. 17 shows that huCAN46G4, huCAN46G19, huCAN46G24 were efficacious at the dose of 0.25 mg/mouse in protecting against lethal toxin B challenge with 100% survival 3 days after the toxin B challenge.

Example 17 Total Human IgG ELISA

The total human IgG ELISA was performed using the Human IgG ELISA Quantitation Set from Bethyl Laboratories (Cat No. E80-104) and following the kit instructions. The ELISA was performed on sera samples collected from mice undergoing Toxin B challenge. The sera was collected from the mice 12 hours after mAb injection, which was 12 hours prior to toxin challenge, and then again at the end of the study, day 4. The time between the first and second sample was 84 hours. The linear rate of decline of detectable circulating mAb was determined by the following calculation:

[(conc. of mAb in serum 12 hours pre-challenge)−(conc. mAb in serum 96 hours post challenge)]/84 hours

Results:

As shown in FIG. 18, the concentrations of mAb were relatively stable over an 84 hour period in the mice injected with either the huCAN46G24 mAb (75 ug & 250 ug), or the rehuCAN46G19 mAb (250 ug). The mice injected with either huCAN46G19 (75 ug & 250 ug) or rehuCAN46G24 (250 ug) lost between 50-75% of detectable circulating mAb. Levels of circulating mAbs in the mice, for all mAbs tested, did not fall below 12 ug/ml after the 4 days post challenge. The mAbs tested were purified from HEK293F cells expressing the Per.C6-based construct.

Example 18 Toxin B Affinity

Affinity of the humanized CAN46 mAb variants and toxin B was measured using biolayer interferometry. Stock antibodies were diluted to 1 mg/ml with PBS and then biotinylated for 30 minutes at room temperature using a commercially-available kit (Fisher Cat No. P121329) using a ratio of 1 mmol biotin/1 mmol Ab. Desalting columns were used to remove excess or unbound biotin. The Octet QKe instrument was equipped with either streptavidin (SA), anti-human Fc (AHC), or anti-mouse Fc (AMC) sensors. The sensors were pre-washed in PBST until a stable baseline was obtained. The mAbs at a concentration of 40 ug/ml were coupled/loaded onto each of the 8 sensors. The sensors were washed again in PBST until a stable baseline was obtained and all unbound mab was removed. The sensors were associated with a dilution series (50 nM to 0 nM) of toxin B and then washed in PBST to assess dissociation of the toxin from each mAb. The Results were analyzed using ForteBio Data Analysis Software to determine the equilibrium dissociation constant (KD) or the strength of binding, the rate at which the mAb:toxin complex forms (kon), and the rate at which the mAb:toxin complex dissociates (kdis).

Results:

Antibodies tested were purified from HEK293F cells expressing the Per.C6-based construct. As shown in FIG. 24, the huCAN46 mAbs had smaller KD values and therefore higher affinity for TcdB when compared to their murine counterparts. Although the rehuCAN46 mAbs did have smaller KD values from their murine counterparts, they did not have significantly different Kdis values. All humanized mAb versions showed similar KD values, 0.15-0.42M, indicating a similar level of high affinity for toxin B. Similar analysis of humanized variants purified from CHOK1 SV cells expressing the CHO-based construct are shown in FIG. 36. Similar affinity constants were obtained for the CAN46G13a variants, while CAN46G24 variants exhibited subnanomolar affinity constants following the humanization protocol.

Example 19 Clostridium difficile Clinical Isolate Toxin B Neutralization Assay with Vero Cells Using the xCELLigence™ Platform Cell Line—

Vero cells are monkey kidney fibroblasts. These cells have been selected since they are highly sensitive to toxin B while relatively resistant to toxin A.

xCELLigence™ Platform

The xCELLigence™ is a real-time label-free cell analysis (RTCA) system based on an electronic impedance cell sensing measurement that evaluates changes in cell characteristics in real-time. Cell growth and cytotoxicity can be detected by monitoring the increase or decrease of a dimensionless parameter called cell index (CI). When adherent cells are cultured within the custom 96-well plate, cell growth characteristics can be monitored in real-time by changes in electrical impedance as measured by the gold electrodes embedded within each well.

The CI measurement is based upon four parameters: 1) cell number, 2) cell size and morphology, 3) cell viability, and 4) cell adhesion. An increase in any one of these parameters leads to an increase in the CI. Conversely, a decrease in any one of these parameters leads to a decrease in CI.

C. Difficile Clinical Isolate Culture Supernatants Preparation

Nine epidemic prevalent clinical isolates and one reference strain (ATCC43255) were selected for toxin B neutralization test. Spore stocks were streaked on brain heart infusion+0.1% taurocholate (BHI-T) plates and cultured at 35° C. in anaerobic chamber for 48 h. Single clones were transferred to 50 ml TY medium and cultured for 4 days. Bacteria cultures were centrifuged and supernates filtered through 0.2 μm filter. Supernatants were stored in 4° C. and cultured in BHI-T plates for 48 h to confirm sterilization. Culture supernatants were diluted with Vero medium to pre-determined concentrations (Table 11) that induces 80-90% cytotoxicity on vero cells.

TABLE 11 C. difficile strains for toxin B neutralization and dilutiong factors for cytotoxicity Dilution factors Description Toxinotype for supernatants ATCC 43255 0 (A⁺B⁺CDI⁻)  1:300,000 C. difficile K-14 0 (A⁺B⁺CDI⁻) 1:1,000 C. difficile Y-2 0 (A⁺B⁺CDI⁻) 1:8,200 C. difficile B1 0 (A⁺B⁺CDI⁻) 1:8,000 C. difficile J9 0 (A⁺B⁺CDI⁻) 1:500  C. difficile BI-6 III (A⁺B⁺CDI⁺)  1:10,000 C. difficile BI-1 III (A⁺B⁺CDI⁺)  1:16,500 C. difficile BI-17 III (A⁺B⁺CDI⁺)  1:10,000 C. difficile CF-2 VIII (A-B+) 1:2,000 C. difficile R23 0 (A⁺B⁺CDI⁻) 1:350 

Vero cells were trypsinized from a T-75 flask and added to a Roche 96-well E-plate® at 7500 cells/well, and incubated about 4 hours at 37° C. During the 4-hr incubation, anti-TcdB mAb dilutions were prepared on a 96-well U-bottom plate. Samples were then mixed with an appropriate dilution of TcdB (0.5-50 ng/mL range, dilution dependant on toxin lot) by repetitive pipetting. The plate is then incubated at 37° C. for about 60 minutes. After completion of initial cell incubation, the cells were overlayed with the toxin/mAb preparation and incubated for a minimum of 72 hours at 37° C. Impedance measurements were taken every 30 minutes throughout the incubation period. This data is plotted in real-time using the xCELLigence™ RTCA software. A single time point representing the optimal time point (either for toxin cytotoxicity or neutralization) was selected. The data from that single time point is used to create a 4-parameter logistic curve for analysis. If sample potency was being determined, the sample curves are constrained against the “reference” sample. Curve constraint is used to constrain the upper/lower asypmtotes, and slope of the curve. This allows for each curve to shift horizontally along the x-axis based upon the curves IC₅₀ value. For potency determination the IC₅₀ value of the standard is divided by the IC50 value of the sample.

Cell Attachment Phase—xCelligence™ Method

This phase included the following steps. (1) Trypsinized cells in source flask. (2) Added 2 mL of trypsin to flask and washed cells to remove traces of media then aspirated. (3) Added 3 mL of trypsin and incubated at 37° C. for approximately 8 minutes. (4) Added 6 mL of assay media to flask. (4) Centrifuged suspended cells at 368×g for 8 minutes. (5) Aspirated supernatant and resuspended cells with 6 mL of assay media. (6) Counted cells and calculated required volume of cells for plating at 7500 cells/well. (7) To a 96 well E-plate added 100 μL of assay media to all wells. (8) Performed background reading on xCelligence. (10) Added 50 μL of 1.5×10⁵ cells/mL suspension to these wells for a final 7500 cells/well seeding density. (11) Incubated plate at room temperature for 20-30 minutes to allow cells to settle evenly. (12) Placed plate in 37° C. incubator with 5% CO₂ overlay 4-5 hours.

Toxin B Preparation:

(1) Prepared Toxin B overlay by diluting primary stock (409.6 μg/mL) to 200 pg/mL (2) Prepared Toxin B for titration by diluting primary stock to 80 ng/mL. (3) Dilutions of primary stock were performed as shown in Table 12.

TABLE 12 TcdB Test Volume of Volume of 10% Sample Concentration TcdB (μL) Medium (μL) Toxin Overlay  (i) 5.43 μg/mL 3 297 (Stock = (ii) 54.3 ng/mL 3 of (i)  297 543 μg/mL) (iii) 200 pg/ml  3 of (ii) 811.5

Sample Preparation:

To test potency, all the monoclonal antibodies were prepared at appropriate concentrations as shown in Table 13.

TABLE 13 Sample Test start Concentration Volume of Volume of 10% Sample (10⁻⁶ M) TcdB (μL) Medium (μL) MDX1388 (Standard; 300 μg/mL 30 120 Medarex anti-TcdB) 1.5 mg/mL hPA-41.1 (1.5 mg/mL) 300 μg/mL 30 120 (Progenics anti-TcdB) HuCAN46G24-2-3 300 μg/mL 18 132 (2.5 mg/mL) rehuCAN46G24 300 μg/mL 36 114 (1.25 mg/ml) Hu CAN46G13a 300 μg/mL 30 120 (1.5 mg/mL) rehuCAN46G13a 300 μg/mL 30 120 (1.5 mg/mL) HuCAN46G19-3-2 300 μg/mL 18 132 (2.5 mg/mL)

Dilution Plate Preparation-xCelligence

The following was performed using a U-bottom 96-well plate: (1) Added 45 μL of assay media to wells B3-H9, and C10-D11. (2) Added 100 μL of media to wells B10-11. (3) Added 50 μL of diluted mAbs (10⁻⁶M or 300 μg/ml)) to corresponding wells B2-H2 as shown below in Table 14. (4) Serially diluted each sample 10-fold by transferring 5 μL from Row 2 and adding to Row 3, mixed and repeated through to Row 9, discarded 5 μl from Row 9. (5) Add 45 μl diluted toxin B (200 pg/ml) to C10, 11; Add 45 μl diluted isolate culture supernatant (Table 11) to D10, 11 (6) Add diluted isolate 1 culture supernatant (Table 11) to wells B2-H9, (7) Plate(s) was shaken on a plate shaker until homogeneous. (8) Incubated at 37° C. with 5% CO₂ for 60 minutes. Table 13 shows the xCelligence dilution plate layout.

TABLE 14 xCelligence Dilution Plate Layout: test of neutralization of C. difficile culture supernatant toxin B. Plate layout: Isolate 1, diluted culture supernatant from selected C. difficile isolates; CC: culture medium control; TC: pure toxin B control (100 pg/ml). 1 2 3 4 5 6 7 8 9 10 11 12 A 10⁺⁶ M 10⁺⁷ M 10⁺⁸ M 10⁺⁹ M 10⁺¹⁰ M 10⁺¹¹ M 10⁺¹² M 10⁺¹³ M B Isolate 1 MDX-B CC CC C hPA-41 TC TC D HuCAN46G24-2-3 isolate isolate E reHuCAN46G24 F HuCAN46G13a G reHuCAN46G13a H HuCAN46G19-3-2

Sample Addition to Cell Plates:

(1) Following completion of incubations, the cell and dilution plates were removed from incubator. (2) Transferred 50 μL of samples from dilution plate to appropriate wells of cell plate. (3) Incubated 72 hours at 37° C. with a 5% CO₂ overlay.

Data Analysis:

(1) Plate data at the 72 hour time point was fit to a 4-parameter logistics (4-PL) curve for each individual sample using Softmax Pro (v.5.4) software. (2) Standard and sample curves were constrained (upper/lower asymptotes, and slope), and the IC₅₀ value of the standard was divided by the IC₅₀ of the sample to determine a potency estimate (when applicable).

Results

FIG. 26 summarizes the EC₅₀ data for each mAb demonstrating the ability of humanized CAN46G24, CAN46G13a, and CAN46G19 variants produced in CHOK1SV cells to neutralize the toxicity of C. difficile clinical isolates. The bar graphs show the EC50 of each mAb against a specific clinical isolate of C. difficile. The isolates include one reference strain (ATCC43255) and 9 representative clinical isolates including three hypervirulent 027 strains (BI-1, BI-6, and BI-17). The EC50 was different for each mAb against each clinical isolate. In general, humanized CAN46G24 and CAN46G19 mAbs neutralized non-NAP1 strains, whereas humanized CAN46G13a mAbs were more effective against NAP1 strains

Example 20 Efficacy of Humanized Toxin B mAbs in a Hamster Gastrointestinal Primary Infection Model with C. difficile B1 Spores

Groups of hamsters received 4 injections of anti-toxin A and anti-toxin B mAbs with either high (50 mg/kg body weight) or low dosages (20 mg/kg bodyweight) each day for four days before infection. On the third day of antibody injection, hamsters were also given 10 mg/kg (bodyweight) of clindamycin to clear gut bacteria flora to enhance C. difficile spore infection. Coincident with the last day of antibody injection, hamsters were intragastrically given 140 B1 spores and clinical signs and survival were recorded twice a day for 22 days, along with the body weights every two days. At day 22 after infection, all surviving hamsters were euthanized and sera collected for anti-toxin antibody levels assayed by Bio-Plex® MAGPIX™ multiplex assay. Refer to Table 15 for full experimental procedure and table 16 for the injections given to each group of hamsters.

The raw data of survival, clinical sign and body weights were analyzed by Graphpad Prism 5 software. Serum was collected prior to antibody injection (Day-3) for all animals and day 22 for all surviving hamsters. Serum specimens were analysed for the injected toxin-specific antibodies by Magplex.

TABLE 15 Experimental Procedure Day Action −8 Receive and acclimatize hamsters. Two hamsters per cage with free access to sterile food and water and exhibit no clinical symptoms of CDI. −3 Administer antitoxins (50 mg/kg each, 200 μl, i.p.) to Group B and C, Administer antitoxins (20 mg/kg each, 200 μl, i.p.) to Group D and E. Administer saline (200 μl, i.p.) to Group A −2 Administer antitoxins (50 mg/kg each, 200 μl, i.p.) to Group B and C, Administer antitoxins (20 mg/kg each, 200 μl, i.p.) to Group D and E. Administer saline (200 μl, i.p.) to Group A −1 Administer antitoxins (50 mg/kg each, 200 μl, i.p.) to Group B and C, Administer antitoxins (20 mg/kg each, 200 μl, i.p.) to Group D and E. Administer saline (200 μl, i.p.) to Group A. Clindamycin (10 mg/kg) administration to all hamsters. 0 Administer antitoxins (50 mg/kg each, 200 μl, i.p.) to Group B and C, Administer antitoxins (20 mg/kg each, 200 μl, i.p.) to Group D and E. Administer saline (200 μl, i.p.) to Grp A. Gavage all hamsters with 140 spores C. difficile strain B1. 1-22 Monitor hamsters twice a day for clinical signs and record clinical scores up to day 21 after infection. Measure body weights every second day of surviving hamsters. Euthanize moribund animals, 22 Terminate experiment and euthanize any surviving animals.

TABLE 16 Group Assignments groups hamster Spores/hamster other A 5 140 No anti-toxins (negative control) B 7 140 CDA1/MDX-1388 50 mg/kg treatment C 7 140 HeCan20G2/HuCAN46G24 50 mg/kg treatment D 8 140 CDA1/MDX-1388 20 mg/kg treatment E 8 140 HeCan20G2/HuCAN46G24 20 mg/kg treatment

Results:

Hamsters were infected with 117CFU/animal of C. difficile B1 spores and were observed twice per day for 22 days. Anti-TcdA and TcdB mAbs, purified from CHOK1 SV cells expressing the CHO-based construct, were administered once a day at 50 mg/kg or 20 mg/kg for −3, −2, and −1 days prior to infection, and 0 days with spores on the day of infection. Verification of B1 spores and viability was performed by serial dilutions of B1 spore inoculums plated on brain heart infusion+0.1% taurocholate (BHI-T) agar and incubated in anaerobic chamber for 48 hours, and confirmed 117 CFU/hamster was administered during infection. FIG. 27 shows the survival data from the hamster primary infection model. Four of five control hamsters (Group A; no treatment) died within 48 h after infection and the fifth one died 96 h after spore administration, indicating infection was established. For the animals treated with antibodies (groups B through E) the final survival rates were variable from 40% to over 80%, which is significantly different from the control group A (without antibody treatment), indicating the protective function of toxin-specific antibody treatment. The survival rates of mAb treated hamsters were found to be dose- and source-dependent. Three of seven hamsters from group B (CDA1/MDX-1388 50 mg/kg) died of infection (one died two days after infection (DAI), another two died 13 DAI) while only one of seven hamsters from group C (HeCan20G2/HuCAN46G24 50 mg/kg) died of infection on day 12 during the experiment period. For lower dosage (20 mg/kg) treatment groups, five of eight animals died in group D (CDA1/MDX-1388) and four of eight hamsters died in group E (HeCan20G2/HuCAN46G24) treated group. Cangene mAbs HECAN20G2 and huCAN46G24 were efficacious at a dose of 50 mg/kg with 100% survival after 12 days and 80% survival after 22 days. This combination was also efficacious at a dose of 20 mg/kg with 100% survival after 16 days and 60% survival after 20 days. Therefore, at equivalent doses, HeCan20G2/HuCAN46G24 treatment led to a higher survival in comparison to CDA1/MDX-1388 treatment, while for the same sourced mAbs, the higher dose was correlated with higher/longer survival rates.

FIG. 28A shows the average body weight (BW) of all groups decreased after infection and recovered during the course of treatment in the primary infection model. The BW of Group B (CDA1/MDX-1388 50 mg/kg) decreased to 55% of the original baseline BW by 13 DAI. In group C (HeCan20G2/HuCAN46G24 50 mg/kg) BW decreased after infection to 73%, started to recover by 8 DAI, reached 87% baseline BW by 16 DAI, and remained stable at about 90%. For 20 mg/kg treatment groups, BW of both group D (CDA1/MDX-1388) and group E (HeCan20G2/HuCAN46G24) decreased to 65% of baseline BW and started to recover thereafter. At the end of experiment, BW of group D animals reached 85% of original weight, while group E reached 80% of the original baseline BW.

FIG. 28B shows the concentration of toxin mAbs in the sera of surviving hamsters on day 22 in the treated groups (groups B, C, D, and E). Post-infection (day 22) hamster serum samples showed anti-Toxin A IgG antibody concentrations between 8.83 μg/mL-308.72 μg/mL. Anti-Toxin B IgG antibody concentrations ranged between 8.92 μg/mL-85.11 μg/mL.

The results from this study indicate that treatment of C. difficile B1-infected hamsters with the combination of humanized HeCan20G2/HuCAN46G24 at both dosage levels (50 mg/1 g and 20 mg/kg) effectively and robustly protected the hamsters from disease, both following infection and subsequent relapse, improving long term survival in comparison to hamsters treated with CDA1/MDX-1388.

Example 21 Characterization of Humanized mAbs Derived from Per.C6 Vector Against C. difficile Clinical Isolates

Concentration of C. difficile Suspensions

C. difficile were selected to represent various strains, including strains known to cause CDI in humans (NAP1 by PFGE). From these strains, C. difficile bacteria were grown from spores in TY broth (200 mL) for 4 days at 35° C. in an anaerobic chamber. Bacterial suspension was pelleted by centrifugation and the supernate was filtered (0.22 μm) to remove any remaining spores and vegetative cells. Supernates were concentrated (Centricon 70 plus) and precipitated with ammonium sulfate. The slurry was mixed by slow stirring 10-12 hours at 4° C. and centrifuged. The resulting pellet was washed 5 times with Tris/NaCl to remove residual ammonium sulfate. The resulting toxins were concentrated by Centricon as above and stored (in Tris/NaCl) at 4-8° C. until tested.

ELISA—Toxin B Determination, Sandwich ELISA

All the C. difficile strains in this study, with the exception of CF2 (A−B+), produce both Toxin A and B (A+B+). A sandwich ELISA was performed to determine concentration of Toxin B in the concentrated supernate derived from clinical isolate strains. Microtiter plates were coated with test articles (various mAb) at 1 μg/mL (16 hrs at 4° C.). After coating, the excess capture mAb was removed and the plates were blocked (5% milk for 1 hr at 37° C.). The blocking reagent was discarded and plates were washed (3× with PBST). Diluted standards and concentrates (in 2.5% milk) were added to the plates and incubated (1 hr at 37° C.). The standard/sample were removed, plates washed and detector Abs (rabbit pAbs to Toxin B) was added (1 hr at 37° C.). The detector was removed, plates washed and conjugate Abs (anti-Rabbit HRP) was added (1 hr at 37° C.). After removal and washing of the conjugate, substrate (TMB) was added and allowed to develop. Reaction was stopped with 1 N H₂SO₄. Plates were read at 450 nm. Analysis was done by SoftMax.

Direct ELISA—Cross-Reactivity ELISA

In addition, direct ELISA was performed to test the cross-reactivity of the humanized CAN46 mAbs with the clinical isolate strains. Microtiter plates were coated with C. difficile concentrates (16 hrs at 4° C.). After coating, the excess concentrate was removed and the plates were blocked (5% milk for 1 hr at 37° C.). The blocking reagent was discarded and plates were washed (3× with PBST). Diluted Abs (rabbit pAbs, mouse and humanized mAbs, in 2.5% milk) were added to the plates and incubated (1 hr at 37° C.). The Abs were removed and plates were washed. The appropriate conjugates were added (anti-rabbit, anti-mouse and anti-human, 1 hr at 37° C.). After removal and washing of the conjugate, substrate (TMB) was added and allowed to develop. Reaction was stopped with 1 N H₂SO₄. Plates were read at 450 nm.

Neutralization Assay—CT26 Cells

CT-26 cells were grown in RPMI-1600 media (with 10% FBS, 37° C., 5% CO₂), plated at 3×10⁴ cells/well and allowed to attach to plates (˜3 hrs). Toxin concentrations/dilutions used were pre-determined during cytotoxicity testing (% viability). Toxins and Ab preparations were mixed (1:1) and allowed to incubate (1 hr, room temperature) After cell attachment, the media from the CT-26 cells plates was removed and 100 μL of each toxin/Ab mixture was added the CT-26 plate. Plates were incubated for ˜48 hrs (37° C., 5% CO₂). After incubation, plates were observed to determine cell rounding. For cell viability, 10 μL/well of WST-1 was added to each well and further incubated (1 hr, 37° C., 5% CO₂). Plates were read at 440 nm and analyzed for % viability by comparison to cells controls

Results: Concentrated toxins from the C. difficile strains were coating on to plates and mAbs bound to the coated toxins. Binding to the coated toxins was defined as follows: High binding=OD>0.800, moderate binding=ODs<0.800 and >0.200, low binding=OD<0.2. FIG. 29 shows that all mAbs showed high binding to the reference strain, ATCC43255. The huCAN46G13a showed the most binding above low levels across the clinical strains tested. In general the antibodies binding to TcdB Fragment 1 showed better binding to the isolated toxins compared to the TcdB Fragment 4 binding mAbs. To standardize these responses, immunoreactivity relative to rabbit pAbs (rpAb) was done as follows: OD of the mAb tested ×100/OD of the rpAb. Both variants (Hu and rehu) for CAN46G24 and CAN46G13a were immunoreactive against the different C difficile toxin B. When similar analysis was performed using sandwich ELISA, toxin B from the different clinical isolates was immunoreactive to varying degrees (FIG. 31). FIG. 31 demonstrates that the huCAN46G13a mAb and the Progenics mAb showed similar binding characteristics to all NAP1 strains tested. The huCAN46G24 mAb and the Medarex mAb showed similar binding characteristics to all strains tested. To demonstrate the relationship between the two ELISA methods, results were analyzed by Pearson correlation. The correlation and significance values for several mAbs are found in Table 17.

TABLE 17 Pearson Correlation between sandwich and cross- reactivity ELISA and associated p values Pearson Statis- Corre- tical lation Corre- Signif- (r) lation p value icance Cangene huCAN46G24 0.93 Strongly 0.00003 Yes mAbs Positive huCAN46G13a 0.41 Positive 0.24 No Comparator hpA41 0.63 Positive 0.04 Yes mAbs <DX-1388 0.94 Strongly 0.00002 Yes Positive r values>0 indicates a positive relationship between results of the two ELISAs. p values≦0.05, indicate that there was statistical significance correlation between the results. For huCAN46G24, hpA41B and MDX-1388, the Pearson correlation is both positive and statistically significant. For huCAN46G13a, the correlation is positive but not statistically significant. Taken together, this suggests that either method provides a relative determination of TcdB:anti-TcdB interactions across the different clinical isolates Since the C. difficile concentrates contain both toxins A&B, when concentrates were used in neutralization experiments, Toxin A mAb (HECAN20G2) was combined with the Toxin B mAbs being investigated, to quench cytotoxic activity of TcdA. Monoclonal Ab concentration were tested from 10 μg/mL to 0.8 μg/mL and % neutralization was calculated at each Ab concentration. To standardize the responses, the average % neutralization between mAb concentrations 5 to 0.16 mg/mL were calculated and used to rank the effectiveness of the mAbs tested. These are illustratively shown in FIG. 30. HECAN20G2/huCAN46G24 neutralized B1, but had reduced protection against NAP1 strains (BI-1, BI-6, BI-17). In contrast, HECAN20G2/huCAN46G13a displayed the reverse trend; reduced protection against the non-NAP1 strain tested (B1) but neutralized BI-1, BI-6 and BI-17. When HECAN20G2 was combined with comparator anti-toxinB candidates (hpA41 or MDX1388), superior neutralization was observed in comparison to the comparator combinations alone (ProA/ProB, MDXA/MDXB).

In FIG. 32, the immunoreactivity to the non-NAP1 strains is presented as a percent relative to a rabbit polyclonal to Toxin B. The 6 mAbs tested showed high immunoreactivity to the ATCC reference strain and low immunoreactivity to strain J9, CF2, R23. Although most mAbs showed weak binding to B1, K14 and Y2, huCAN46G13a showed high immunoreactivity to B1 and Y2 and moderate immunoreactivity to K14

In FIG. 33, the immunoreactivity to the NAP1 strains is presented as a percent relative to a rabbit polyclonal to Toxin B. The huCAN46G13a mAb and the Progenics mAb showed high immunoreactivity to all strains tested. The other mAbs tested, huCAN46G24, rehuCAN46G24, rehuCAN46G13a, and the Medarex mAb showed weak immunoreactivity to all strains tested.

In summary, the 4 mAbs tested showed similar binding characteristics to all strains tested. The binding to toxin from C. difficile strain J9 was the most diverse across the mAbs with huCAN46G13a showing the weakest binding.

Example 22 Production of Humanized C difficile Toxin mAbs

For production of humanized C difficile toxin mAbs, individual IgG sequences encoding for heavy and light chains are co-expressed in vectors under the control of promoter, including a Kozak/HAVT20 leader sequence and terminator. Intron/exon sequences were added to the 3′ end of each variable sequence followed by a double stop codon to signal the end of transcript translation. For the kappa light chain, this included one intron/exon (constant exon). For the heavy chain, this included four sets of introns/exons (CH1, CH2 and CH3 constant exons). Introns were included in the sequence to allow for eukaryotic processing of the mRNA transcript. Expression constructs could be used for transient expression in adapted mammalian cell lines (HEK293F, CHO-S, CHOK1SV, Per.C6) for transient transfection by lipofectamine. The expression vectors also included appropriate selectable markers for each expression system to enable stable expression in mammalian cell culture using electroporation. For experiments requiring comparative analysis, mAbs were synthesized from published sequences ecoding CDA1 (3D8 kappa chain GenBank accession number DJ444525; heavy chain GenBank accession number CS483823), MDX-1388 (124-152 kappa chain accession number CS483846, heavy chain CS483842), and hpA41 and hpA50 sequences from international publication WO2011130650(A2). Heavy and light chains were synthesized and cloned into full length IgG1 vectors for expression in CHO-K1SV, HEK293F, Per.C6 systems. The IgG1 mAbs were expressed and purified as outlined above and served as positive controls for anti-TcdA activity (CDA1, specificity against TcdA fragment 4) for HeCAN20G2, or anti-TcdB activity with specificity against fragment 1 (hpA41) for CAN46G13a or fragment 4 (MDX-1388) for CAN46G24, CAN46G19, and CAN46G4. Following transient transfection, supernatants were decanted, filtered (0.22 um) and concentrated using a stir-cell concentrator and a 30 kDa membrane. Concentrate was filtered (0.22 um) prior to purification. For stable transfections, clones were screened and isolated for assessment in batch and fed-batch growth. IgG, concentrated and filtered supernatants were purified on Protein G columns, buffer exchanged and concentrated. For final concentrates, protein content was determined by BCA assay or with an Octet QKe instrument equipped with Protein A biosensors against standard curves for human IgG equivalents.

Results: Table 18, shows representative expression titers for huCAN46G24 and huCAN46G13a in transient and stable transfections. From these means material was supplied to conduct characterization, in vitro and in vivo analysis

TABLE 18 Representative expression titers normalized to mg/L in transient and stable expression lines (BOG = batch overgrowth, FOG = fed batch overgrowth). Stable (CDCHO-v8 media) C difficile anti-TcdB Transient BOG FOG CAN46G24 7.4 647.8 4,129.5 CAN46G13a 6.3 8.8 117.6

While specific aspects of the invention have been described and illustrated, such aspects should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims. All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. 

What is claimed is:
 1. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 110, 111, and 112, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 102, 103 and 104, respectively.
 2. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 126, 127 and 128, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 118, 119 and 120, respectively.
 3. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 142, 143 and 144, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 134, 135 and 136, respectively.
 4. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 206, 207 and 208, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 198, 199 and 200, respectively.
 5. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 222, 223 and 224, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 214, 215 and 216, respectively.
 6. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain region and a light chain region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 238, 239 and 240, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 230, 231 and 232, respectively.
 7. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 238, 239 and 240, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 230, 231 and 232, respectively.
 8. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain region, wherein the heavy chain variable region comprises amino acid sequences about 80% to about 100% homologous to SEQ ID NO. 710 and the light chain variable region comprises amino acid sequences about 80% to about 100% homologous to SEQ ID NO.
 708. 9. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody, or antigen-binding portion thereof, binds to C. difficile toxin B, and wherein the dissociation constant (K_(D)) of the antibody, or antigen-binding portion thereof, is less than about 1×10⁻⁸ M.
 10. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof is humanized or chimeric.
 11. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 76, 77 and 78, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 68, 69 and 70, respectively.
 12. The antibody or antigen-binding portion thereof of claim 12, wherein the heavy chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 75, and wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO:
 67. 13. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 44, 45 and 46, respectively, and wherein the light chain variable region comprises three CDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 36, 37 and 38, respectively.
 14. The antibody or antigen-binding portion thereof of claim 13, wherein the heavy chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 43, and wherein the light chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO:
 35. 15. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof is selected from the group consisting of: (a) a whole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab′)2; and (e) a disulfide linked Fv.
 16. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof comprises at least one constant domain selected from the group consisting of, (a) an IgG constant domain, (b) an IgA constant domain, (c) IgD constant domain, and (d) IgE constant domain.
 17. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof binds to fragment 1 of the C. difficile toxin B.
 18. The antibody or antigen-binding portion thereof of claim 4, wherein the antibody or antigen-binding portion thereof binds to fragment 4 of the C. difficile toxin B.
 19. An isolated monoclonal antibody or an antigen-binding portion thereof, that binds to C. difficile toxin B and comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 109, 125, 141, 157, 173, 189, 205, 221 and 237 and comprises a light chain variable region, wherein the light chain variable region having an amino acid sequence about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NOs: 101, 117, 133, 149, 165, 181, 197, 213 and
 229. 20. An isolated monoclonal antibody or an antigen-binding portion thereof, that binds to C. difficile toxin B and comprises a heavy chain variable region, wherein the heavy chain variable region comprises a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NOs: 389, 405, 421, 437, 453, 469, 485, 501, 517, 533, 549, 565, 571, 587, 603, 619, 635, 651 and 709, and wherein the light chain variable region comprises a nucleic acid sequence about 80% to about 100% homologous to the nucleic acid sequence set forth in SEQ ID NOs: 381, 397, 413, 429, 445, 461, 477, 493, 509, 525, 541, 557, 563, 579, 595, 611, 627, 643 and
 707. 21. The isolated monoclonal antibody, or an antigen-binding portion thereof, of claim 1, wherein, in an in vivo toxin B challenge experiment, when the antibody or an antigen-binding portion thereof, is administered to a mammal at a dosage ranging from about 8 mg/kg body weight to about 13 mg/kg body weight about 24 hours before the mammal is exposed to greater than about 75 ng of C. difficile toxin B, the chance of survival for the mammal is greater than about 80% within about 4 days after the exposure to toxin B.
 22. The isolated monoclonal antibody, or an antigen-binding portion thereof of claim 1, wherein the antibody, or antigen-binding portion thereof, at a concentration ranging from about 25 μg/ml to about 100 μg/ml, neutralizes greater than about 40% of about 5 ng/ml C. difficile toxin B in an in vitro neutralization assay.
 23. A cell comprising the nucleic acids set forth in claim
 24. 24. The cell of claim 18, wherein the cell is a bacterial cell.
 25. The cell of claim 18, wherein the cell is a eukaryotic cell.
 26. The cell of claim 18, wherein the cell is a mammalian cell.
 27. The cell of claim 21, wherein the cell is COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, SP2/0, HeLa, Per.C6, myeloma or lymphoma cells.
 28. A composition comprising the antibody or antigen-binding portion thereof of claim 1 and at least one pharmaceutically acceptable carrier.
 29. A method of preventing or treating C. difficile-associated disease comprising administering to a subject an effective amount of the antibody or antigen-binding portion thereof of claim
 1. 30. The method of claim 28, wherein the antibody or antigen-binding portion thereof is administered intravenously, subcutaneously, intramuscularly or transdermally.
 31. The method of claim 28, further comprising the step of administering to the subject a second agent.
 32. The method of claim 31, wherein the second agent is an antibiotic.
 33. The method of claim 32, wherein the antibiotic is vancomycin, metronidazole, or fidaxomicin.
 34. The method of claim 31, wherein the second agent is an antibody or antigen-binding portion thereof that binds C. difficile toxin A. 